lc–ms/ms analysis of bath salts and other novel psychoactive...

LC–MS/MS Analysis of Bath Salts and Other

Novel Psychoactive Substances

Combining New and Traditional Ionization

Methods for MS

Automated 2D-HPLC–MS for the

Characterization of Protein Modifi cations

Volume 16 Number 1 March 2018

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5 Current Trends in Mass Spectrometry March 2018 chromatographyonl ine .com

Articles

Liquid Chromatography Tandem Mass Spectrometry Method for Novel Psychoactive Substances: Kratom and Synthetic Cathinones in Urine 6

Debashish Roy, Oneka T. Cummings, Erin C. Strickland, Allyson L. Mellinger, Christa L. Colyer, and Gregory L. McIntire

A novel “dilute-and-shoot” LC–MS/MS method is described for the analysis of “bath salts” sold as “legal” highs, including mitragynine and nine synthetic cathinones, in urine.

Combining Novel and Traditional Ionization Methods for Mass Spectrometryfor More Comprehensive Analyses 12

Sarah Trimpin, Santosh Karki, Darrell D. Marshall, Ellen D. Inutan, Anil K. Meher, Sara Madarshahian,

Madeline A. Fenner, and Charles N. McEwen

Novel ionization processes provide gas-phase ions of a wide variety of materials using MS. These simple and sensitive methods operate from solution or a solid matrix. Both manual and automated platforms are described that allow rapid switching between the ionization methods of MAI, SAI, vSAI, and conventional ESI.

Rapid Online Reduction and Characterization of Protein Modifications Using Fully Automated Two-Dimensional High Performance Liquid Chromatography–Mass Spectrometry 18

Anja Bathke, Denis Klemm, Christoph Gstöttner, Christian Bell, and Robert Kopf

A fully automated process for online peak fractionation and reduction of therapeutic antibodies with subsequent QTOF-MS characterization is presented. The technique is based on state-of-the-art 2D-HPLC technology coupled with additional HPLC modules via a dedicated software macro.

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Ad Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Application Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Cover image courtesy of KAMONRAT / shutterstock.

March 2018

chromatographyonl ine .com6 Current Trends in Mass Spectrometry March 2018

Debashish Roy, Oneka T. Cummings, Erin C. Strickland, Allyson L. Mellinger, Christa L. Colyer, and Gregory L. McIntire

A novel “dilute-and-shoot” method is described for the analysis of “bath salts” sold as “legal” highs,

including mitragynine and nine synthetic cathinones. The cycle time for this method is 2.5 min. No

sample clean-up or extraction was performed. This assay monitors two tandem mass spectrometry

(MS/MS) transitions for each of the following 10 analytes: alpha-PVP, butylone, ethylone, MDPV,

mephedrone, methcathinone, methedrone, methylone, naphyrone, and mitragynine; and two

internal standards: methylone D3 and alpha-PVP D8. This straightforward “dilute-and-shoot” liquid

chromatography (LC)–MS/MS method produces acceptable limit of detection (LOD) and limit of

quantitation (LOQ) limits without costly extraction and time-consuming concentration protocols.

Liquid Chromatography Tandem

Mass Spectrometry Method for

Novel Psychoactive Substances:

Kratom and Synthetic Cathinones

in Urine

Cathinone is a monoamine alkaloid found in the Khat plant (Catha edulis) (1). Khat is a shrub grown in eastern Africa and the southern Arabian Peninsula, which can have mild

stimulant effects when the leaves are chewed (2). Cathinone is also a beta ketone analog of amphetamine and as such is reported to cause sympathomimetic effects as well as psychoactive effects common with amphetamine and amphetamine derivatives such as MDMA (“Ecstasy”) and MDA (“Eve”) (3). Chewing of the Khat plant has been linked to increased risk of heart-related dis-eases and duodenal ulcers (4). Synthetic variants of cathinone, commonly referred to as “bath salts” or as “novel psychoactive substances” (NPS) by public health personnel, are stronger and more dangerous than their natural counterpart (5). The first syn-thetic derivatives of cathinone were reported during the 1920s (6-8). These initial derivatives were designed for medical purposes; however, most were withdrawn as a result of abuse and obvious dependency among users. Interestingly, bupropion (Wellbutrin) is a “synthetic cathinone” and is widely used as an antidepressant and anti-anxiety medicine (2).

More recently, synthetic cathinones (NPS) have expanded as popular drugs of abuse. Their ready availability in the local drug market and so-called head shops make them easy “legal” highs. These active agents are sold under various names such as pesticides and insect repellants. They are often labeled as “not for human consumption,” to avoid regulatory control. However, the use of bath salts has significantly increased in recent years with poison control centers in the United States reporting 304 calls related to bath salts in 2010 and 1782 calls in the first four months of 2011 (9,10). It is believed that the in-crease in their use is a result of their availability, lack of proper methods for drug testing, and media coverage.

Mitragynine is a naturally occurring substance found in the leaves of the kratom (Mitragyna speciosa) plant, a mem-ber of the coffee family found in Southeast Asia, Indochina, and Malaysia (11). This “legal” high is readily available and is often used to mitigate pain, aid in opiate withdrawal, and for recreational purposes (12). This natural drug is regulated in a number of countries including Malaysia but

chromatographyonl ine .com March 2018 Current Trends in Mass Spectrometry 7

not currently scheduled in the United States (13).

Urine drug testing is a common means of monitoring for the use of these drugs. While forensic laboratories often have ac-cess to blood, urine, and other tissues to test in post-mortem subjects, pain man-agement laboratories tend to focus on urine or oral fluids because of the ease of sampling and minimal stability require-ments. As well as ease of sample collec-tion, the sample preparation required is comparatively easier for urine than other patient specimens, for example, blood. Often such preparation consists of sample matrix removal using solid-phase extraction (SPE); however, reports of “dilute-and-shoot” testing confirm that direct analysis without sample prepara-tion is possible for some analytes (14). In this study, a unique “dilute-and-shoot” liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the analysis of nine synthetic cathinones and mitrag-ynine (Kratom) in patient urine samples. This method is ideal for a high-through-put production environment because it has a modest 2.5-min cycle time and re-quires low sample preparation time and cost while still preserving sensitivity and specificity of the quantitative analytical result. Above all, this method achieved complete chromatographic resolution of the isobars, ethylone and butylone, both of which are analyzed in this method, by lowering column temperature to 20 °C and cutting flow rate to 0.4 mL/min to maintain system pressure.

Materials and

Methods of Analysis

Reagents

All reference standard compounds, including internal standards, were purchased from Cerilliant. Solvents, including methanol, acetonitrile, and formic acid, were purchased from VWR. Normal drug-free urine was obtained from UTAK.

Preparation of Calibration Standards

Aliquots of normal drug-free urine were fortified with the reference standards at concentrations of 25 ng/mL, 250 ng/mL, and 1000 ng/mL as calibrators. All samples, calibrators, and controls were

diluted 5× with the internal standard solution (0.5 μg/mL methylone D3 and alpha-PVP D8) by adding 400 μL of in-ternal standard to 100 μL of sample.

Instrumentation

All analyses were performed via LC–MS/MS on Acquity TQD UPLC/MS/MS system (Waters). A 2.1 × 50 mm, 1.7-μm Acquity UPLC BEH C18 analyti-cal column (Waters) was used for chro-matographic separations. The run time

for this method was 2.0 min with a total cycle time of 2.5 min. No sample extrac-tion or clean-up was required before LC–MS/MS analysis. Two transitions were monitored for each of 10 analytes (alpha-PVP, butylone, ethylone, MDPV, mephedrone, d,l-methcathinone, me-thedrone, methylone, mitragynine, and naphyrone), plus two internal standards (methylone D3 and alpha-PVP D8), as shown in Table I. Elution solvents used in the gradient were 0.1% formic acid

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Table I: Ion fragmentation transitions of analytes and internal standards

Analyte M+1 Cone Voltage (V)Quantifi er Ion (1st transition)

Collision Energy (V)

Qualifi er Ion (2nd transition)


Alpha-PVP 232.00 26 76.97 38 104.98 26

Butylone 222.07 22 174.13 18 72.03 22

Ethylone 222.10 20 174.13 20 146.10 26

MDPV 276.12 36 126.07 26 135.00 30

Mephedrone 178.08 22 145.02 18 119.06 22

D,L-Methcathinone 164.03 24 130.96 20 104.99 22

Methedrone 194.08 22 161.05 18 58.01 14

Methylone 208.06 14 160.03 16 132.00 28

Mitragynine 399.00 42 110.05 30 159.00 58

Naphyrone 282.15 20 141.01 22 211.14 20

Internal Standard M+1 Cone Voltage (V) Quantifi er IonCollision Energy

(V)Qualifi er Ion


Alpha-PVP D8 240.08 34 76.97 44 91.01 24

Methylone D3 211.08 20 163.04 16 135.08 30

in 10% methanol (mobile phase A) and 0.1% formic acid in 50:50 methanol– acetonitrile (mobile phase B), with a flow rate of 0.4 mL/min (Table II).

Method Validation

The method was validated by examin-ing various parameters such as limits, linearity, precision and accuracy (P&A: Ten replicates of each of three concentra-tions each day for three days with coef-ficient of variance [%CV] of each set of P&A point replicates each day and across all P&A days falling within +/- 15%), interferences, and matrix effects. Drugs of abuse (benzoylecgonine, THC-A, am-phetamine, methamphetamine, MDMA, MDEA, MDA, and phentermine) and therapeutic drugs (oxazepam, morphine, imipramine, buprenorphine, fentanyl, meprobamate, methadone, tramadol, gabapentin, pregabalin, and tapentadol) were tested as possible sources of inter-

ferences. Patient samples were evaluated by comparing the quantitative results of a previously used method with those of the new method as described herein. Since mitragynine and alpha-PVP were not evaluated in the previous method, a com-parison was not possible. The previous method was developed and validated as described here but lacked critical analytes, for example, mitragynine and alpha-PVP. This method has not been reported in the literature. Detailed discussion of these validation parameters can be found in a paper by Enders and McIntire (14).

The tolerance for all analytes at each concentration level was ± 25%. All daily calibration curves had correlation coef-ficient values (r2) greater than or equal to 0.99. The matrix blanks following the highest calibrator points did not show any signs of carryover, that is, no re-sponse greater than 50% of the limit of quantitation (LOQ).

Two mass transitions were selected for each of the analytes following infusion studies of pure standards dissolved in matrix. The first transition served as the quantifier ion transition and the second transition served as a qualifier ion transi-tion (Table I). The ratio of the areas of the quantifier ion transitions for the analyte of interest and the internal standard (IS) were used to obtain the relative response by plotting the area ratio of the analyte-to-IS versus analyte concentration. The area of the second ion transition of the analyte was used to generate a specific relative ratio to the area of the quanti-fier peak. This generates a compound specific “ion ratio” for each analyte that together with retention time helps to confirm that the peak results from the compound of interest.

ResultsThis method generated calibration data with consistent peak areas and ion ratios for all analytes and internal standards. Daily three-point calibration curves were completed with correlation coef-ficient values (r2) of at least 0.99 for all analytes. Consistent accuracies of 100 ± 25% were obtained for all calibration points, negative and positive controls, as well as points generated daily for both calibration and controls. The limit of quantification (LOQ) was determined to be 25 ng/mL, which was sufficient to

Table II: Liquid chromatography gradient parameters

Step Time (min) Flow Rate (mL/min) %A %B

1 0.00

0.400

80 20

2 0.20 80 20

3 1.80 20 80

4 1.81 2 98

5 1.86 2 98

6 2.00 80 20

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Table III: Validation data obtained from the “dilute-and-shoot” method for all analytes included in this assay

Precision and Accuracy Matrix Interference

CarryoverAvg. Conc.

ng/mL (N = 5)

Avg. % Target (N = 30) Avg. % CV (N = 30)% Matrix

EffectInterfering compounds75 ng/

mL300 ng/

mL750 ng/

mL75 ng/

mL300 ng/mL 750 ng/mL

Alpha-PVP 5.3 100.2 95.9 96.7 3.4 1.3 1.1 3.97 None

Butylone 0.6 97.7 95.0 97.3 4.5 1.9 3.5 -20.34 Ethylone

D,L Methcathinone 0.0 111.2 97.1 97.0 3.0 0.5 1.2 -7.95 None

Ethylone 1.9 100.8 97.3 97.1 1.6 1.6 0.9 -2.50 Butylone

MDPV 3.1 112.8 108.3 102.2 3.5 4.4 3.2 20.45

None

Mephedrone 1.3 102.1 94.1 96.1 1.4 1.7 2.5 21.22

Methedrone 0.0 109.8 102.1 98.0 1.9 0.9 1.2 6.31

Methylone 2.5 102.7 96.6 97.5 1.6 0.8 1.3 -16.91

Mitragynine 9.3 105.4 101.2 104.5 0.4 3.0 4.8 -18.86

Naphyrone 8.5 105.1 103.1 104.4 1.5 0.6 5.7 -56.43

LOQ was 25 ng/mL for all analytes. ULOL was set at 1000 ng/mL for all analytes

Figure 1: Overlaid chromatogram of 10 analytes determined in this assay. The chromatogram

was obtained after injecting a sample at the limit of quantification (LOQ) of 25 ng/mL. Internal

standards (IS) are not shown.

achieve consistent precision and accura-cies as well as acceptable ion ratios, while the upper limits of linearity and of car-ryover (ULOL, ULOC) were 1000 ng/mL. The chromatogram of all analytes shown in Figure 1 provides clear evidence that there is good chromatographic separation and resolution of all analytes assessed in this assay even at the LOQ. Furthermore, Figure 2 shows the separation achieved for the ethylone and butylone isobars, while Table III shows a summary of the validation results.

Conclusions This work documents the successful development and validation of an im-proved LC–MS/MS method for routine, high-throughput analysis of alpha-PVP, butylone, ethylone, MDPV, mephedrone, d,l-methcathinone, methedrone, methy-lone, mitragynine, and naphyrone, using methylone D3 and alpha-PVP D8 inter-nal standards. During the initial stages of method development, the column tem-perature was held at 50 °C with a flow rate of 0.8 mL/min, which required a

much longer 4.0 min cycle time for com-plete resolution of all analytes of interest. This was especially true for the isobars butylone and ethylone, which did not exhibit robust separation even with the longer elution time. To ensure complete resolution (< 10% peak height overlap) of these two isobars while still achieving the desired 2.5 min cycle time, the column temperature was lowered to 20 °C and the flow rate was decreased to 0.4 mL/min. Traditional chromatography para-digms indicate that higher temperatures lead to better resolution (15). However, the unique combination of column, sol-vents, analytes, and flow rates used here responded to lowering the temperature for improved resolution. Lowering the column temperature coupled with de-creasing the gradient flow rates proved vital to ensure that appropriate system pressures were maintained while facilitat-ing the appropriate degree of separation of the isomers, ethylone and butylone.

The synthetic cathinone compounds of interest were successfully analyzed in urine samples by this “dilute-and-shoot” LC–MS/MS method. This particular method is robust and useful because it reduces analysis time and does not incur additional costs for up-front sample preparation while still providing accurate results. As there were no interferences observed from the major drugs of abuse and therapeutic drugs, this method is an excellent tool for the clinical, medicinal, and toxicology communities, especially

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Table IV: Positive NPS data from a 14-month period of testing

Analyte Mitragynine Alpha-PVP Methcathinone Butylone Methylone Ethylone MDPV

Avg 1849.5 2134.3 142.8 5389.4 102.0 2299.0 92.0

Std Dev 4582.5 4275.0 156.8 10273.5 42.0 540.2

Median 589.0 258.5 75.0 220.0 84.0 2299.0

Max 37615.0 19521.0 698.0 34070.0 150.0 2681.0

Min 26.0 29.0 25.0 29.0 72.0 1917.0

n 81 24 25 14 3 2 1

Values in all rows are reported with units of ng/mL, with the exception of the bottom row, which is reported as the number of samples yielding a positive result. Naphyrone, mephedrone, and methedrone were not detected.

in high sample volume settings. Results achieved with this method

are summarized in Table IV where posi-tive data from over a year of testing are summarized. Mitragynine was the most prevalent component in the tested urine samples, while alpha-PVP and meth-cathinone were close seconds. Although tests for these analytes are positive for just a small fraction of all pain medica-tion monitoring samples, these results are important to both physicians and patients in their ongoing treatments for chronic pain, substance abuse, and mental illness. For example, patient ages associated with at least one positive bath salt result ranged from 19 to 72 years old, with equal posi-tivity rates across all age groups. Lastly, the overwhelming positivity of mitragy-nine in this group more than likely re-flects the legality of this natural product in many states in the USA, while attempts to schedule this agent at the federal level have been delayed by consumer activist protests (13).

Any method that tests for illicit synthetic substances must be up-dated frequently to keep up with the changes on “the street” that result in novel pharmaceutical substances being consumed with known and unknown effects. However, this method offers advantages in terms of cost and time and has been shown to be capable of detecting many patient positives when implemented for large numbers of samples.

AcknowledgmentsThe authors would like to thank the National Science Foundation (NSF-GOALI Award 1611072) for financial support of this work, together with Wake Forest University and Ameritox.

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Debashish Roy and Allyson L.

Mellinger are with the Chemistry

Department at Wake Forest University

in Winston-Salem, North Carolina and

Ameritox, LLC, in Greensboro, North

Carolina. Oneka T. Cummings,

Erin C. Strickland, and Gregory

L. McIntire are with Ameritox.

Christa L. Colyer is with the

Chemistry Department at Wake Forest

University. Direct correspondence to:

[email protected] ◾

Figure 2: Chromatogram showing the separation of the isomers ethylone and butylone.


Sarah Trimpin, Santosh Karki, Darrell D. Marshall, Ellen D. Inutan, Anil K. Meher, Sara Madarshahian, Madeline A. Fenner, and Charles N. McEwen

Novel ionization processes provide gas-phase ions of a wide variety of materials using mass

spectrometry (MS). In the case of inlet ionization, the sub-atmospheric pressure region of the

mass spectrometer becomes the “ion source.” These simple and sensitive methods operate

from solution or a solid matrix. Matrix-assisted ionization mass spectrometry (MAI-MS) uses

a solid matrix spontaneously producing gas-phase analyte ions upon exposure to sub-atmo-

spheric pressure without the need for any external energy input. The solvent-based method

of solvent-assisted ionization mass spectrometry (SAI-MS) ionizes certain classes of com-

pounds more efficiently than electrospray ionization mass spectrometry (ESI-MS), and voltage

solvent-assisted ionization mass spectrometry (vSAI-MS) is a highly sensitive combination

of ESI and SAI. Direct analysis of tissue, fluids, including whole blood and urine, biological

extracts, reaction mixtures, and buffered, salty solutions detecting drugs, metabolites, lipids,

and proteins, simultaneously, demonstrates the advantages of the multi-ionization concept

for obtaining more comprehensive analyses from 384-well plates and, if desired, hyphenated

with liquid chromatography (LC), further simplifying complexity. The methodology devel-

oped for inlet ionization allows rapid switching between MAI, SAI, vSAI, and conventional ESI

increasing the comprehensiveness of mass measurements at equal or better mass and drift

time resolution.

Combining Novel and Traditional

Ionization Methods for Mass

Spectrometry for More

Comprehensive Analyses

Advancing chemical analysis through fundamental research and applying this knowledge to improve analyses in biological sciences will impact areas such

as medical diagnostics (1). Mass spectrometry (MS), because of its specificity, sensitivity, dynamic range, wide applica-bility, and potential for high-throughput, is widely used in biological analyses. MS directly provides atomic and molecu-lar composition information of heterogeneous bulk samples and surfaces, important for educated decisions on materials composition associated with quality, performance, safety,

and more recently disease diagnostics. Integral to advances in MS are developments for transferring molecules into the gas phase regardless of size or volatility. As was demonstrated with electrospray ionization (ESI) and matrix-assisted laser desorption–ionization (MALDI), successful new ionization methods can have an impact on science far beyond anything envisioned in their early discovery, as witnessed by the award of a portion of the Nobel Prize in Chemistry to John Fenn and Koichi Tanaka in 2002. ESI and MALDI have strengths and weaknesses relative to analytical needs, which is the case


with all ionization methods used with MS. Mass spectrometer manufacturers have recognized the need for multiple ionization methods by designing ion sources capable of ESI, atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoion-ization (APPI) as well as offering AP-MALDI and intermediate pressure MALDI on some of these instruments. Ambient ionization methods such as at-mospheric solids analysis probe (ASAP) (2), desorption electrospray ionization (DESI) (3), and direct analysis in real time (DART) (4) have also become popular. In this article, we describe new ionization techniques and manual and automated approaches that allow rapid switching between these ioniza-tion techniques as well as with ESI. The combination of methods allows inter-facing with liquid separation methods, as well as direct analysis of samples, and increases the likelihood that im-portant compounds will not be missed because of ion suppression or poor ion-ization efficiency.

New means of transferring small, large, volatile, and nonvolatile com-pounds from the solid or liquid state directly into gas-phase ions were dis-covered through fundamental research and developed into methods providing exceptional sensitivity and simplicity (5). The initial discovery began using laser ablation of common MALDI matrices at AP, but instead of singly charged ions expected of MALDI,

multiply charged ions expected of ESI were observed (6,7). Because the mass spectra looked like ESI but used laser ablation, the method was termed la-serspray ionization (LSI) (Figure 1a). Fundamental studies led to the un-anticipated finding that the laser is unnecessary because analyte ions are formed when neutral matrix–analyte enters a heated inlet tube of a mass spectrometer (Figure 1b) (8,9). Further studies showed that the matrix could be the solvent used to dissolve the ana-lyte (Figure 1c). Use of a solid matrix was termed matrix-assisted ioniza-tion (MAI) (10) and use of a solution was called solvent-assisted ionization (SAI) (11,12). While the method of introduction of the matrix sample into the heated inlet tube, which be-comes the ion source, differs in LSI, MAI, and SAI, the results relative to charge states are the same, and thus the representative term inlet ioniza-tion (13,14). However, the heated inlet tube was found not to be necessary provided that heat is applied through laser absorption by the matrix, or astonishingly, without any added ex-ternal energy by using an appropriate volatile matrix (15,16). In all cases, multiply protonated analyte ions are formed from compounds having mul-tiple basic sites, such as peptides and proteins, just as in ESI. Voltages, ob-structions, or additives can improve ionization in these new ionization processes (9,16–21).

Experimental

Materials Drugs (verapamil, fexofenadine, eryth-romycin, azithromycin, and hydroxy-chloroquine), peptides (leucine-en-kephalin, bradykinin, and angiotensin I), proteins (bovine insulin, ubiquitin, and cytochrome c), LB broth “Len-nox”, and 3-nitrobenzonitrile (3-NBN) were purchased from Sigma Aldrich. High performance liquid chromatog-raphy-grade water, methanol, acetoni-trile, and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific Inc. and absolute ethanol from Decon laboratories. All chemicals were used without further purification.

Sample Preparation

Stock solutions of drugs were prepared in ethanol (verapamil and azithromy-cin), 1:1 acetonitrile–water (erythro-mycin and hydroxychloroquine), and DMSO (fexofenadine). For peptides and small proteins, stock solutions of 1 mg/mL were prepared in water. Di-lutions were made in 50:50 methanol–water (drugs) and in water (peptides and small proteins) to a final con-centration of 5 μM, unless otherwise noted in the text. For MAI using the automation platform, protein samples were prepared in 1:1 (v:v) methanol–water solutions and 100 mg of 3-NBN matrix was prepared in 3 mL of 3:1 (v:v) acetonitrile–water solution. For the bacteria extraction protocol, 50 mL of bacterial strains was grown to sta-tionary phase from overnight starter cultures at 37 °C and 200 rpm on LB media. The cultures were pelleted at 4 °C for 15 min at 4000× g and washed 3× times with cold water. The pellets were resuspended in double distilled (dd) water and lysed with an ultra-son-icator by alternating 10 s on/off cycle while on ice. Cell debris was separated by centrifugation at 16,000g, 4 °C for 15 min and 1.0 mL of the supernatant collected with two methanol–water ex-tractions and dried on a biodryer, fro-zen, and lyophilized. The samples were then resuspended in ethanol. For the MALDI source, the matrix–analyte mixture was prepared in 1:3 (v:v) and 1 μL of the mixture was spotted on the sample plate.

Figure 1: Graphical representations of inlet ionization methods: (a) LSI in transmission geometry,

(b) MAI vacuum cleaner, (c) SAI liquid junction pen, and (d) DAII (droplet assisted inlet ionization).


Instrumentation and Data Acquisition Thermo Fisher Scientif ic Orbitrap Exactive and Waters Synapt G2S mass spectrometers were used. The Orbi-trap Exactive was operated at 100,000 mass resolut ion (mass-to-charge [m/z] 200, 50%). The Synapt G2S in-strument was operated in mobility-time-of-f light (TOF), sensitivity, and positive-ion mode detection. MAI-MS using the MSTM multifunctional au-tomation platform was operated on the Synapt G2S mass spectrometer. The MSTM automation platform re-placed the Waters ion source housing and was mounted on the instrument using a specially designed f lange (22). For the automation measurements of proteins, 0.2 μL of 3-NBN and 0.1 μL of the protein solution was aspirated using a fused-silica capillary (internal diameter [i.d.] ~100 μm), and upon dispensing at the tip of the fused silica capillary allowed to dry for a few seconds prior to injection into a modified inlet tube for mass spec-tral analysis. For the bacterial strain, mass spectra were obtained using ESI by spraying 1.0 μL of the extract with 3 kV applied on the source, and MAI using 0.1 μL of the extract combined with 0.1 μL of the 3-NBN matrix so-lution, air dried and then transferred into the modified inlet. The source temperature was set at 80 °C. For MAI-MS using the MALDI source, no laser and low energy settings were used: sample plate 0–10 V, extraction 10 V, hexapole 10 V, and aperture “0” 5 V.

Results and Discussion

For those mass spectrometers that provide a heated inlet tube, ioniza-tion is readily achieved for any of the inlet ionization methods after the

source is removed for access to the inlet aperture of the mass spectrom-eter. Examples are shown in Figure 1, with and without the use of a laser, in which various surfaces were sampled using, for example, a pen through which fused-silica tubing was held in place to form a liquid junction (Figure 1c). The solution, after contacting a surface, was sucked into the section of fused silica tubing, the exit end of which was inside the heated inlet tube and, therefore, at sub-AP (22) similar to previous studies (23,24).

Some of the small-molecule MAI matrix compounds, especially those which spontaneously produce analyte ions when exposed to sub-AP, have no labile hydrogen atoms and thus can-not directly donate protons (25). A common feature of all matrices which spontaneously produce analyte ions when subjected to sub-AP is that they sublime under the conditions of the experiment (26). Without sublima-tion, no ions are observed from these matrices. It is therefore surprising that such a fundamental chemical process as sublimation might stil l harbor secrets. Protic solvents, such as water or methanol, present in at least a few percent, enhance ioniza-tion in MAI, and especially of a non-volatile analyte. However, drying the matrix sample prior to insertion into sub-AP does not hinder the ionization process, thereby demonstrating that ionization does not occur through liquid droplets. Studies to date sug-gest solid matrix particles are the engine of charge separation and the solvent the proton source. This means that ESI-like charge states are pro-duced not only from charged liquid droplets as in ESI, but also from solid charged particles. On the other hand,

SAI, in which the matrix is the solvent used to dissolve the analyte, produces ions most effectively in a heated inlet tube linking AP and the vacuum of the mass analyzer without applica-tion of a voltage, and must produce ions through highly charged liquid droplets similar to ESI. However, SAI and ESI ionize many compounds with different efficiencies. A hybrid method that combines the attributes of ESI and SAI, and termed voltage SAI (vSAI), where a voltage is applied to a solution introduced directly into a heated inlet tube through fused-sil-ica tubing, enhances sensitivity and combines attributes of ESI and SAI. The potential for high-throughput and automated analyses were previ-ously demonstrated for MAI and SAI (27,28), and tissue imaging applica-tions for LSI (29–31).

The inlet ionization methods of MAI, SAI, and vSAI offer highly sensitive di-rect ionization methods using solid ma-trices like MALDI, but without a laser, a solution like ESI, but without the need of a voltage, and the solution method with a voltage that combines attributes of ESI and SAI. Each of these ionization meth-ods offers compound-specific ionization efficiencies. For example, SAI has been shown to ionize certain steroids more ef-ficiently than ESI (32) and MAI has low ionization efficiency for compounds that typically ionize by metal cation attach-ment and high efficiency for ionization of compound classes such as drugs and peptides. MAI, using 3-NBN as a matrix, ionizes typical background compounds with a low efficiency, which can be ad-vantageous for trace analyses as was demonstrated for a sample of the peptide angiotensin I consuming just 5 femto-moles to produce a clean mass spectrum (33). This attribute makes MAI ideal for the analysis of urine, blood, and tissue samples for drugs and their metabo-lites, and proteins from buffered and detergent conditions, as demonstrated for the Ebola virus protein and bacte-riorhodopsin, a membrane protein, as well as others (34–37). Metal cation ad-ducts or other undesired attachments, for example matrix molecules, produced using MALDI or ESI are not observed with MAI matrices. Negative ions are

Table I: Comparison of ion intensities in an LC–MS study for erythromycin (MW 733), hydroxychloroquine (MW 335), and leucine-enkephalin (MW 555) using ESI, vSAI, and SAI using orbital trap MS detection

MoleculesMolecular

Weight (g/mol)

Volume Used (μL)

Concentration (ppm)

Ion Intensity

ESI vSAI SAI

Erythromycin 733 1 0.26 6.7e4 6.9e5 2.05e5

Hydroxychloroquine 335 1 0.002 7.8e3 6.2e5 3.1e6

Leucine-enkephalin 555 1 0. 38 7.9e4 6.5e5 4.0e3


spontaneously produced allowing, for example, detection of ganglioside and other lipids, such as cardiolipins, di-rectly from a simple mitochondria ex-tract, and carbohydrate conjugates as singly and multiply deprotonated ions with minimal fragmentation, which is often not the case using ESI or MALDI

(38,39). Combining these new ionization methods with ESI in a single, easy-to-use platform gives the user the best chance to rapidly analyze compounds easily missed if only ESI, only MALDI, or only a single inlet ionization method are used for the analysis.

With the manual platform, the liq-

uid introduction methods of ESI, SAI, and vSAI can be interfaced with an liquid chromatography (LC) system for LC–MS analyses. Ion intensities, in an LC–MS study, using each of the liquid introduction methods with the drugs, erythromycin (MW 733) and hydroxychloroquine (MW 335), and the peptide, leucine-enkephalin (MW 555), were determined for injection of 1 μL of solution (Table I). The same ion intensity trends were observed at higher concentrations, vividly show-ing differences in ionization efficien-cies between the ionization methods. Additional supporting information relative to ionization efficiencies of ESI, SAI, and vSAI using LC–MS has been provided in a previous publica-tion (19).

As might be expected, there are also differences in mass spectra ob-tained by direct ionization using MAI, MALDI, and ESI. The differ-ences are best seen when compar-ing complex mixtures. One example is the comparison of ESI and MAI using the automated system which is capable of analyzing 384-well mi-crotiter plates or surfaces, for ex-ample glass plates, using ESI, MAI, SAI, or vSAI, just as with the manual system. Three strains of E. coli bac-teria were grown, lysed, extracted in ethanol, and five replicates of each extract were acquired by MS using MAI, with the 3-NBN matrix in 3:1 acetonitrile–water, as well as with ESI directly from the ethanol extract. The results for the m/z range 400–800 of MV1184 E. coli in ethanol are shown in Figure 2. Some of the differences observed are ionization by Na+ ad-duction in ESI versus protonation in MAI, for example m/z 726 and 704, while other ions, for example m/z 749 and 765 in ESI and ions around m/z 637 in MAI, are not observed in both mass spectra. Because protonated ions fragment more readily, the MAI mass spectrum shows mass differences of m/z 141 (m/z 690 to 549 and m/z 704 to 563), likely representing loss of the polar head group from a glycerophos-pholipid. Thus, some of the contents of this extract as viewed by these two ionization methods would seem to be

Figure 2: Comparison of MV1184 E. coli bacterial extract in 100% ethanol using the automated

platform (22) and ionized by (a) ESI-MS and (b) MAI-MS on a Synapt G2S instrument.

Figure 3: (a) Total ion chromatogram of 50 acquisitions and (b) MAI mass spectra of protein solutions

using 3-NBN: (1) bovine insulin (MW 5733), (2) cytochrome c (MW 12384), and (3) ubiquitin

(MW 8560). The mass spectra were acquired using the automated platform (22) on a Synapt G2S

instrument.

ES1027022_LCGCCTMS0318_015.pgs 03.06.2018 22:58 ADV blackyellowmagentacyan


different. An example of an automated ac-

quisition of three proteins multiple times is shown in Figure 3 using MAI with 3-NBN as a matrix. Note that the mass spectra look nearly identi-cal in charge states to ESI, but here ionization is from a solid rather than a liquid matrix. MAI has some dis-tinct advantages relative to ESI. For example, simply using a different ma-trix provides different selectivity al-lowing ionization to be tailored to the problem, similar to MALDI. The most studied MAI matrices of the over 40 discovered so far are 3-NBN, 1,2-dicy-anobenzene, 2-bromo-2-hydroxy-1,3-propandiol, and 2-methyl-2-hydroxy-1,3-propandiol, two of which have no labile hydrogen atoms, and yet all matrices produce singly or multiply protonated analyte ions similar to ESI. MAI, as noted earlier, does not require a heated inlet tube if the proper ma-trix is used. In fact, an intermediate

pressure MALDI source can be used for analysis of multiple samples with-out the need for a laser (40,41). Figure 4 shows the analyses of six samples within 50 s in one row of a MALDI plate with samples spaced every other well. This approach has considerable potential if the time to introduce the plate into the ionization region can be greatly reduced from the current ca. 2 min. Interestingly, MAI has been reported to have equal or better mass and ion mobility resolution than ESI and MALDI on the same and other mass spectrometers (5,27).

The SAI method of ionization also has advantages over conventional ion-ization for certain types of analyses. As with any new discoveries, the util-ity becomes most apparent when oth-ers begin to apply the new technology in innovative ways. For example, the Johnston group at the University of Delaware introduced droplet assisted inlet ionization (Figure 1d), which,

for some compounds, provides up to four-orders of magnitude better ion sensitivity than ESI for direct aerosol analysis (42). Professor Eberlin at the University of Texas at Austin reported on an “MS pen” device for detection of cancerous tissue during surgery using solvents as the matrix in inlet ioniza-tion (43). There are a number of other examples where solvent matrices were used without application of high volt-age as required with ESI, including zero volt paper spray (44–46). Imag-ing applications and MS/MS of multi-ply charged ions directly from surfaces have also been adapted by the group of Professor Li (University of Wis-consin) (47). MAI matrices have been used with ambient ionization sources with success (48,49). Professor Murray’s group at Louisiana State University constructed a pulsed valve MAI device achieving 300 times more intense signal with 3-NBN than with 2,5-dihydroxy-acetophenone, a common MALDI ma-trix (50). The width and breadth of use of the new ionization processes indicate f lexibility and broadness of the initial inventions and findings.

ConclusionsFundamental studies relative to the new ionization technologies and methods for sample introduction continue to develop. The simplicity of ionization processes that need only exposure to the vacuum of the mass spectrometer or a heated inlet tube suggests applications where costs and sim-plicity are most critical. Developments in automated sample introduction with ever increasing speed and high sensitivity, and without carryover between samples or instrument contamination are being de-veloped. Areas of application are antici-pated to be in the general areas of clinical, pharmaceutical, and medical diagnostics applications, because of simplicity, speed, and robustness, but also field portable mass spectrometers because of the low pumping capacity requirements of MAI. The one thing that is clear is that the re-search so far has reshaped how one must think about ionization in a fundamental and applied fashion.

AcknowledgmentNSF CHE-1411376 (to ST) and NSF

Figure 4: MAI fast analysis of six sample spots in 50 s: (a) Photograph of the sample plate with six spots

(on each row) of the matrix–analyte mixture using 3-NBN as matrix, (b) total ion chromatogram, and

(c) representative MAI mass spectra of the analytes from drugs (verapamil, MW 454; fexofenadine,

MW 501; azithromycin, MW 748), peptides (bradykinin, MW 1059; angiotensin I, MW 1296), and small

protein (bovine insulin, MW 5733). Data acquired using the commercial MALDI source of a Synapt

G2S instrument.

ES1027021_LCGCCTMS0318_016.pgs 03.06.2018 23:01 ADV blackyellowmagentacyan


STTR Phase II 1556043 (to CNM) are gratefully acknowledged. Any opin-ions, f indings, and conclusions or recommendations expressed in this material are those of the author and do not necessarily ref lect the views of the National Science Foundation.

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Am. Soc. Mass Spectrom. 27, 1591–

1596 (2016).

(34) E.D. Inutan, J. Wager-Miller, S.B. Na-

rayan, K. Mackie, and S. Trimpin, Int.

J. Ion Mobility Spectrom. 16, 145–159

(2013).

(35) D.D. Marshall, E.D. Inutan, B. Wang,

C.W. Liu, S. Thawoos, J. Wager-Miller,

K. Mackie, and S. Trimpin, Proteomics

16, 1695–1706 (2016).

(36) S. Trimpin, S. Thawoos, C.D. Foley, D.W.

Woodall, J. Li, E.D. Inutan, and P.M.

Stemmer, Methods 104, 63–68 (2016).

(37) S. Thawoos, J. Wager-Miller, E.D. Inu-

tan, Z.J. Devereaux, C.D. Foley, Y.-H.

Ahn, K. Mackie, P.M. Stemmer, and S.

Trimpin, "Rapid Analysis of Proteins

on High-Resolution Mass Spectrome-

ters using Matrix-Assisted Ionization,"

paper presented at the 64th ASMS

Conference on Mass Spectrometry

and Allied Topics, San Antonio, Texas,

2016.

(38) C. Reynolds, J. DeLeeuw, C. Corinne

Lutomski, T. Sanderson, K. Przyklenk,

and S. Trimpin, "Matrix-Assisted

Ionization-Mass Spectrometry En-

ables Cardiolipin Characterization

Directly From Intact Mitochondrial

Membranes," paper presented at

the 63rd ASMS Conference on Mass

Spectrometry and Allied Topics, St.

Louis, Missouri, 2015.

(39) B. Wang, G. Liao, Z. Guo, and S.

Trimpin, "Characterization of Carbo-

hydrate-Monophosphoryl Lipid Con-

jugate Cancer Vaccine Candidates

using Matrix Assisted Ionization

Vacuum Mass Spectrometry," paper

presented at the 62nd ASMS Con-

ference on Mass Spectrometry and

Allied Topics, Baltimore, Maryland,

2014.

(Continued on page 28)

www.chromatographyonl ine .com18 Current Trends in Mass Spectrometry March 2018

Anja Bathke, Denis Klemm, Christoph Gstöttner, Christian Bell, and Robert Kopf

Characterization of protein modifications is an essential aspect of biopharmaceutical development.

Traditionally, the characterization process of chromatographic peaks involves manual, larger-scale

fractionation to obtain a sufficient amount of material for further analytical studies. This article

presents a fully automated process for online peak fractionation and reduction of therapeutic anti-

bodies with subsequent quadrupole time-of-flight mass spectrometry (QTOF-MS) characterization.

This innovative technique significantly accelerates MS peak characterization compared to traditional

approaches and avoids the risk of unintended modifications of the variants as a result of the isola-

tion process, for example, deamidation during storage of isoforms. This approach considerably

reduces the required sample amount and can be used for the characterization of product-related

impurities during early stage development.

Rapid Online Reduction and

Characterization of Protein

Modifications Using Fully

Automated Two-Dimensional

High Performance Liquid

Chromatography–Mass

Spectrometry

Monoclonal antibodies (mAbs) are highly complex recombinant proteins used successfully as thera-peutics in many diseases. These immunoglobulins

are large tetrameric glycoproteins with a molecular weight of 150 kDa. Monoclonal antibodies for therapeutic use are produced in genetically modified mammalian cells, for ex-ample, Chinese hamster ovary cells. During the expression, process- and product-related side products, such as glyco-sylation variants, glycation, and mispairing for CrossMabs,

are generated. Additional modifications can occur during downstream processing and storage, including deamida-tion, isomerization, oxidation, and truncation (1–3). Aspar-tic acid or asparagine residues in the light and heavy chain are known to form succinimide under mildly acidic condi-tions. Succinimide further isomerizes to isoaspartic acid or back to aspartic acid under neutral or alkaline conditions. Hotspots for aspartic acid isomerization and asparagine deamidation are the motifs DG, NT, and NG (4–7).

www.chromatographyonl ine .com March 2018 Current Trends in Mass Spectrometry 19

These structural a lterations or modifications lead to charge and size heterogeneity. For example, high-mo-lecular-weight species, such as aggre-gates of mAbs, have been correlated to a high safety risk because of their immunogenicity and inf luence on pharmacokinetics and efficacy (8). The formation of isoaspartic acid from a succinimide reaction may lead to a loss of biological activity (6,9). Most of

these modifications can be monitored by the commonly used separation techniques, ion-exchange chromatog-raphy (IEC) or size-exclusion chro-matography (SEC), and, consequently, these methods are commonly used in biopharmaceutical quality control (QC) strategies.

Characterization of charge and size variants is required to generate the product knowledge that forms

the basis for critical quality attribute (CQA) assessment. The former is com-monly performed using off line frac-tionation followed by mass spectrome-try (MS) analysis of reduced protein in combination with enzymatic peptide maps. Although MS-friendly mobile phases for SEC separations have been reported recently (10,11), most buffers used in an IEC or SEC system are not compatible with MS. Therefore, an

Table I: First dimension IEC separation conditions for tested mAbs

Fab 1 Antibody A

Column 4 × 250 mm, 10-μm ProPac WCX-10 Column 4 × 250 mm, 10-μm MabPac SCX-10 BioLC

Flow rate (mL/min) 1.0 Flow rate (mL/min) 1.0

Temperature (°C) 40 Temperature (°C) 40

Buffer A 10 mM Sodium phosphate, pH 7.5 Buffer A 10 mM HEPES, pH 7.7

Buffer B 100 mM NaCl in Buffer A Buffer B 10 mM HEPES, 1 M NaCl, pH 7.7

Gradient

Time (min) Buffer B (%)

Gradient


0 3

2 3 0 0

27.2 45 46 25

28 100 47 100

33 100 49 100

34 3 50 0

40 3 56 0

Antibody B Antibody C

Column 4 × 250 mm, 10-μm ProPac WCX-10 Column 4 × 250 mm, 10-μm ProPac WCX-10

Flow rate (mL/min) 0.8 Flow rate (mL/min) 1.0

Temperature (°C) 25 Temperature (°C) 25

Buffer A 10 mM Sodium phosphate, pH 7.5 Buffer A5 mM Tris, 5 mM Piperazin, 5mM H3PO4,

pH 6.0

Buffer B 100 mM NaCl in Buffer A Buffer B 5 mM Tris, 5 mM Piperazin, 5mM H3PO4,

30 mM NaCl, pH 10.0

Gradient


Gradient


0 15 0 0

30 55 4 0

35 55 10 31

36 100 70 76

44 100 71 100

45 15 76 100

55 1577 0

96 0


off line fractionation with subsequent up-concentration and buffer exchange step has to be performed. This pro-

cedure is very time-consuming and requires considerable amounts of iso-lated material (12), thereby often pro-

hibiting peak characterization during early development. During the prepa-ration, the isolated fraction is exposed

Table II: Second dimension gradient program and valve events

Time (min)Flow Rate (mL/min)

Column Temperature (°C)

MP A(0.1% FA in PWA) (%)

MP B (0.1% FA in acetonitrile) (%)

0.02 M DTT (%)

Valve Phase

0 0.50 70 99 1 0 1 Trapping

5.00 0.50 70 99 1 0 Trapping

5.01 0.056 70 0 0 100 2 Reduction

10.0 0.056 90 0 0 100 Reduction

10.01 0.5 90 99 1 0 2 Wash

17.0 0.5 90 99 1 0 Wash

17.01 0.5 90 99 1 0 3 Separation

23.00 0.5 90 72 28 0 Separation

25.00 0.5 90 68 32 0 Separation

32.00 0.5 90 0 100 0 Wash

37.00 0.5 90 0 100 0 Wash

38.00 0.5 70 99 1 0 3 Reequilibr.

39.5 0.5 70 99 1 0 3 Reequilibr.

Figure 1: Flowchart online reduction. (a) 1D: Cutting of Peak 1 in Deck A, 2D: idle, 3D: re-equilibration of reversed-phase column. (b) 1D: Cutting of further peaks

in Deck B, 2D: Peak 1 trapped on reversed-phase column, 3D: idle. (c) 1D: Cutting of further peaks in Deck B, 2D: idle, 3D: reduction of Peak 1. (d) 1D: Cutting of

further peaks in Deck B, 2D: gradient separation of reduced Peak 1, 3D: idle.

(a)

(c) (d)

(b)

1D Quart. Pump 1D Quart. Pump

IEC

HPLC column

IEC

HPLC column

HPLC column

UV Detector6 Loops Deck A

6 Loops Deck B

RP-HPLC

Valve 1

Waste WasteWaste Waste

TOF MS

Valve 2HPLC column

RP-HPLC

Valve 2

Valve 3TOF MS

Valve 3

Waste


6 Loops Deck B

Valve 1

Waste

2D Binary Pump 2D Binary Pump

3D Binary Pump

1D Quart. Pump

IEC

HPLC column

HPLC column


6 Loops Deck B

RP-HPLC

Valve 1

Waste Waste

TOF MS

Valve 2

Valve 3

Waste

2D Binary Pump

3D Binary Pump

1D Quart. Pump

IEC

HPLC column

HPLC column


6 Loops Deck B

RP-HPLC

Valve 1

Waste Waste

TOF MS

Valve 2

Valve 3

Waste

2D Binary Pump

3D Binary Pump

3D Binary Pump


to stress, which adds the risk of un-intended modifications from sample preparation. To accurately determine the identification of the samples, fur-ther processing is performed including reduction, alkylation, and digestion steps (12–14).

During conventional characteriza-tion of mAbs, whole antibody samples were reduced and digested by dithio-treitol (DTT) or immunoglobulin-de-grading enzyme from Streptococcus pyogenes (IdeS). The fragments were

then separated by IEC and reversed phase in the two-dimensional liq-uid chromatography (2D-LC) setup using comprehensive mode (15,16). By using this approach, variants of a mAb sample can be characterized but not assigned to specific peaks. The scope of this work was the direct character-ization of mAb variant peaks observed in IEC or SEC QC) routine methods in a fully automated workflow. Several attempts were made to establish semi- or full automation for this workf low

using fraction samplers or custom-made systems (17,18). However, most of those solutions were either not fully automated or very complex to handle and set up (17). Tran et al. performed all steps of a peak characterization including an IEC separation, online reduction, and online digestion with subsequent mass analysis via elec-trospray ionization time-of-f light (ESI-TOF) for smaller proteins with a customized capillary chromatographic system using a stop-f low technique.

Figure 2: (a) Total ion chromatogram of the reduced antibody A: The peaks marked with a black arrow show the two light chains. Peaks with orange arrows

represent the two different heavy chains of the antibody. Mass analysis of antibody A light chain. (b) and (c) Deconvoluted spectra of the two different light

chains of the antibody, with molecular weight 21,957 Da and 23,617 Da. (d) and (e) Deconvoluted spectra of the two different heavy chains of the antibody, with

molecular weight 50,635 Da and 77,247 Da.

Inte

ns.

x10

7

Inte

ns.

x1

06

Inte

ns.

x1

04

Inte

ns.

x1

06

6

4

2

0

2.0

1.25

1.00

0.75

0.50

0.25

0.00

1.5

23617 21957

Theo. mass23617

Theo. mass21957

10000 20000 2000030000 3000040000

40000

4000050000

50000

50635

6000080000 120000100000

77247

6000070000

50000

1.0

0.5

0.0

Inte

ns.

x1

04

4

1.25

1.00

0.75

0.50

0.25

0.00

3

2

1

0

5 6 7 8 9 10 11 13

Time (min)

Time (min) Time (min)

Time (min) Time (min)

12

(a)

(b)

Theo. mass50635

(d)Theo. mass

77247

(e)

(c)


However, a fully automated proce-dure allowing use of routine analytical methods common for quality control strategies has been lacking (18).

This article describes a fully au-tomated online reduction 2D-HPLC quadrupole time-of-f light (QTOF)-MS setup that allows rapid charac-terization of protein modifications with a multiple heart-cut valve system for automated peak fractionation. In contrast to earlier work, our process provides not only online reduction

of trapped peaks but also a reversed-phase separation of the reduced pro-teins before entering the MS system. The developed system uses standard HPLC components and allows the use of the established QC methods for larger proteins (mAbs) in the first dimension without any adaptations. Peaks of interest observed in routine QC measurements can be directly characterized without the need for re-conformation runs. The use of a multiple heart-cut module allows for

seamless analysis in the second di-mension without the need to stop the f low. The system is easy to program with commercially available software. This article also discusses the benefits of an online approach versus standard offline peak characterization in detail.

Materials and Methods

Chemicals

Acetonitrile HPLC gradient grade, 2-(N-morpholino) ethanesulfonic acid monohydrate (MES), MES so-

Figure 3: (a) IEC chromatogram of first dimension separation of antibody B. The unknown peak is marked with a black arrow. (b) and (c) Deconvoluted MS

spectrum antibody B, only the heavy chain is shown. (b) Reduced main peak as a reference. (c) MS spectrum of the heavy chain of the fractionated and online

reduced basic peak. The succinimide versions are marked with an asterisk.

mA

U140

Inte

ns.

x1

04

3

Inte

ns.

x1

04

4

3

2

1

0

2

1

0

50550 50600

50583 50601

5068850709

50745 50763

5090850926

50650 50700 50750 50800 50850 50900

50550 50600 50650 50700 50750 50800 50850 50900

50925

50601

50764

+MS, 7.0-7.3min, Deconvoluted (MaxEnt, 740.58-1650.64, 0.1, 50000), Smoothed (1.00,1,GA)


G0

G0

G1

G1

G2

G2

(a)

(b)

(c)

120

100

80

60

40

20

0

-20

10 12 14 16 18 20 22 24 min

Time (min)

m/z

m/z


dium salt, 4-(2-hydroxyethyl)-1-pi-perazineethanesulfonic acid (HEPES), dithiotreitol (DTT), sodium hydroxide (NaOH), and hydrochloric acid (HCL) were purchased from Merck KGaA. A 50% formic acid (FA) solution and phosphoric acid were obtained from Fluka (Sigma-Aldrich Chemie GmbH). Tris(hydroxymethyl)aminomethane

(TRIS), piperazine, disodium hydro-gen phosphate, sodium dihydrogen phosphate , Tris(2-carbox yet hyl)phosphine hydrochloride (TCEP), and sodium chloride were purchased from Sigma-Aldrich. Isopropyl alco-hol (IPA) was acquired from Sigma-Aldrich. Potassium chloride, dipo-tassium hydrogen phosphate, and

potassium dihydrogen phosphate were ordered from Acros Organics. Purified water was used from a Milli-Q water Advantage A1-system (Merck KGaA).

Instrumentation

Experiments for online reduction of mAbs were performed on an Agilent 1290 Infinity 2D–LC system. The

Figure 4: (a) IEC chromatogram of first dimension separation of antibody C. The unknown peak is marked with a black arrow. (b) and (c) Deconvoluted MS

spectrum antibody C, only the heavy chain is shown. (a) Reduced main peak with the expected distribution of the glyco variants G0, G1, and G2. (b) Reduced

acid peak.

(a)

(b)

(c)

mA

U

175

150

125

100

75

50

25

15 20 25 30 35 40 45 min

0

Inte

ns.

x1

04

6

Inte

ns.

x1

04

4

3

2

1

0

49200 49400 49600 49800 50000 50200

502355013150072

49969

49807

50400

49645

4

2

049200 49400

49416

49645

49808

49765

49800

49969

50000

50075

50200 5040049600

G0

G1 / G0 with glycation G2 / G1 with glycation

G2 with glycationG0

G1

G2

+MS, 8.0-8.2min, Deconvoluted (MaxEnt, 1026.50-1467.86, 0.1, 50000)

+MS, 8.0-8.2min, Deconvoluted (MaxEnt, 723.80-1195.19, 0.1, 50000)

Time (min)

m/z

m/z


standard system itself consists of the following modules: 1260 quaternary pump (G5611A), 1290 binary pump (G4220A), 1260 Bio ALS autosampler (G5667A), 1290 thermostat (G1330B), 1290 column oven (G1316C) contain-ing a six-port, two-position valve (G4231B), and two 1260 ultraviolet (UV) detectors (G1314F). The 2D in-terface module consists of a 1290 eight-port, two-position valve (G4236A) and two valve decks for multi heart-cut sampling with 6 × 40 μL loops each (G4242A multiple heart-cutting

kit). To this standard system a third binary pump (G4220A) and a second column oven (G1316A) containing a six-port, two-position valve (G4231B) was added to enable the online reduc-tion of antibodies. For MS detection, a Bruker Impact II ESI QTOF system (0211 G203) was used. The 2D-LC sys-tem was connected via a fused silica capillary with a diameter of 75 μm to the MS system. OpenLab CDS Chem-station (Agilent) was used to control the 2D-LC system. HyStar Software (Bruker) was used to control and ac-

quire data from the MS system. A start signal for each individual 2D run was triggered by a customized software macro by means of a contact closure board. The signal of the 2D UV detec-tor was fed into the Bruker software by an analog-to-digital (A/D) interface net ADC LC 421n (SCPA GmbH).

Starting with injection of the sam-ple and separation on the first col-umn, peaks of interest were fraction-ated online and stored in one of the 40-μL loops. During fractionation of the first dimension one peak after the

Figure 5: (a) IEC chromatogram of first dimension separation of Fab 1 highlighting the peak of interest. (b) and (c) Deconvoluted mass spectra, only the light

chain is shown: (b) Reduced main peak as a reference and (c) fractioned and online reduced acidic peak of the stressed Fab 1 material.

mA

U

15 16 17 18 19 20 21

700

600

500

400

300

200

100

0

-100

(a)

(b)

(c)

Inte

ns.

x10

6

Inte

ns.

x10

5

3

2

1

0

1.0

0.8

0.6

0.4

0.2

0.0

23800 23850 23900 23950 24000

24004

24020

24081

24004

24050 24100 24150 24200 24250

23800 23850 23900 23950 24000 24050 24100 24150 24200 24250



Time (min)

m/z

m/z


other was injected onto the second di-mension where it was either reduced and separated, or separated and then analyzed in the MS system. The pro-cess from injection of the sample until analysis in the MS system was fully automated by a “Valve Event Plugin” Macro from ANGI (Gesellschaft für angewandte Informatik). Each time a fraction was injected into the second dimension the third pump was started again and a start signal was sent to the MS system. In this way, a separate MS data file is generated for each frac-tion. Another advantage of the macro is that a valve in the column oven can be switched time dependent so that no reducing agent or salt from the first dimension IEC or SEC enters the MS system. In the setup for online reduc-tion the second dimension UV detec-tor was excluded to get sharper peaks and a good separation in the MS total ion chromatogram (TIC).

HPLC Operating Conditions

Sample PreparationThree different mAbs and one anti-gen-binding fragment (Fab) were used as a sample for online reduction. For online reduction, the mAb can be in-jected directly without any dilution or preparation.

First Dimension SeparationIEC of mAbs was performed accord-ing to conditions listed in Table I. The columns used were from Thermo Sci-entific.

Second Dimension SeparationTrapping: The cut fractions were di-rected to a 2.1 × 50 mm, 1.7-μm Ac-quity BEH C4 reversed phase column (Waters Corporation) and trapped. Mobile phase A was 0.1% FA in puri-fied water (PWA) and mobile phase B was 0.1% FA in acetonitrile. The pump was set to deliver 99% mobile phase A and 1% mobile phase B at a flow rate of 0.5 mL/min. The column temperature was set to 70 °C.

Online ReductionThe loaded reversed phase trapping column was f lushed with the reduc-tion solution at a f low rate of 56 μL/

min for 5 min. The reduction solu-tion was prepared by either dissolving 0.1543 mg DTT or 0.2502 mg TCEP in 50 mL tris solution pH 7.5 ending up in a final concentration of 0.02 M DTT or TCEP. The reduction solutions were freshly prepared each day.

SeparationThe trapped and reduced peaks were finally separated by applying the gradient conditions as described in Table II.

Process FlowThe complete characterization of a peak of interest consists of four steps (see Figure 1). After cutting the first peak of interest in Deck A, subse-quent peaks were sampled in Deck B. Immediately after cutting, peak 1 was f lushed to the reversed phase column where it is trapped and desalted. By switching valve 2, the trapped peak was reduced by pumping reducing re-agent via the 3D pump. After reduc-tion, valve 2 and valve 3 were switched simultaneously and the reduced mAb chains were separated with a reversed phase gradient and detected by MS.

MS Operation Conditions

For detection of reduced chains as well as material that has not been reduced, a Bruker Impact II system was used. The detection range was set between 300 and 2500 m/z. The end plate off-set was 500 volt and dry temperature was set to 220 °C. Calibration was per-formed with 0.005% phosphoric acid.

Results and Discussion

Development of an

On-Column Reduction Step

Complete on-column reduction of mAb samples was achieved after a 5-min flush with either 0.02 M DTT or 0.02 M TCEP (data not shown). Longer reduction times were also evaluated without any improvement. Based on this observation no further optimiza-tion was deemed necessary. To verify the designed setup with the 5-min f low through of the reducing agent, a CrossMab (19) antibody (Antibody A) with one extended Fab arm on one side (2+1 CrossMab [20]) was used.

For IEC separation conditions refer to Table I. The size of the whole antibody is approximately 194 kDa without ac-counting for additional glycosylation. This antibody is built of two different heavy chains (HC) with a mass of ap-proximately 77 kDa and 51 kDa and two different light chains (LC) with 22 kDa and 24 kDa; the chain with 24 kDa is present two times for each an-tibody. Figure 2a shows a TIC of the antibodies main peak (0.38 μg of pro-tein for each fraction) after reduction. Major peaks could be assigned to the respective HC and LC chains.

Figure 2b and 2c depict the decon-voluted masses of all heavy and light chain fractions after online reduction. Other peak spectra showed compara-ble spectra.

Four different chains of the an-tibody A were detected after online reduction with excellent signal inten-sity (Figure 2). All interchain disul-fide bridges as well as all intrachain disulfide bridges were reduced. The Fc glycosylation of the heavy chains was detected with different glycoforms. The detected masses corresponded to the theoretical masses based on the amino acid sequence of the four dif-ferent chains (Figure 2). In addition, the intact antibody A (without reduc-tion) was analyzed using 2D-LC–MS and the theoretical mass was observed (data not shown).

The developed system was used to analyze unknown peaks in stressed samples of three different antibodies and one Fab Fragment (Fab 1). Anti-body B is a standard IgG1 antibody (Trastuzumab), whereas antibody A and C have extended Fab arms on one side and therefore a higher molecular weight than typical IgGs.

Characterization of a Low Abun-

dance Basic IEC Peak in Antibody B

The IEC chromatogram of antibody B shows a low abundance peak in the basic region (Figure 3a). For IEC separation conditions refer to Table I. A 40-μL sample of the peak of inter-est was extracted (0.24 μg of protein for each fraction) in the first dimen-sion and transferred to the second dimension reversed phase column for


on-column protein reduction. The re-duced species were then eluted using an acetonitrile gradient and analyzed by QTOF-MS. The obtained deconvo-luted mass spectrum is presented in Figure 3b and 3c.

The peak with a mass of 50,601 rep-resents the G0 glycovariant, whereas 50,763 and 50,926 are the G1 and G2 variants (7) of the non-modified heavy chain. The deconvoluted spectrum of the basic peak displays significantly increased signals with a difference of 17 ± 1 Da for all three glycoforms. This difference, combined with the reten-

tion time shift to the basic region of the IEC, suggests isomerization of as-partic acid to succinimide in the heavy chain of the mAb. This conclusion is further supported by data already pub-lished (21). The already present shoul-ders in the main peak spectrum at the same position are method-induced ar-tifacts and represent the loss of water. The same effect can be seen in the spectrum of the basic peak.


dance Acidic IEC Peak in Antibody C

In a second experiment an acidic peak

of an IEC separation of antibody C was analyzed (see Figure 4a). For separa-tion conditions refer to Table I. A 40-μL fraction of the peak of interest (0.54 μg of protein for each fraction) was sampled, reduced, and analyzed by QTOF-MS. The main peak was an-alyzed as a reference. The results are presented in Figure 4b and 4c.

The main peak shows a normal distribution of the glyco variants, with the most abundant var iant being G0, fol lowed by G1 and G2 (Figure 4b). In the spectrum of the analyzed acidic IEC peak (Figure

Figure 6: (a) Workflow for manual peak characterization. (b) Flow chart of an online peak characterization. Different peaks of interest from a LC (IEC or SEC)

chromatogram are fractionated and directly analyzed reduced or none reduced in the MS system.

(a)

(b)

10 X 60minutes

Fractionationmultiple (˜10)

times

Concentration+ Buffer

exchange

Re-injection /Purity + Content

Check

Offlinereduction and

alkylation

Online reduction

2D-HPLC / MS

Online fractionation - total amount of

LC-MSMeasurement of

reduced antobody

LC-MS Measurementof reduced antobody

LC-MSMeasurementof intact mass

LC-MSMeasurement of

intact mass

-time: 190 minutes - > appr. 3h

-protein material: 300 μg antibody

-plastic material: no Centricons

Offline fractionation - totalamount of

-time: 1640 minutes - > appr. 27.5h

-protein material: 1500 μg antibody

-plastic material: 5 Centricons

Online

fractionation

Offline

fractionation

Stress forantibody

480minutes

5 X 60minutes

60 minutes

150 minutes

5 X 20 minutes

2 X 60 minutes

5 X 20 minutes

5 X 20 minutes

5 X 20 minutes


4c), G0 was dramatically reduced, while the G2 peak was significantly increased. This can be explained by glycation of the heavy chain. Glyca-tion is the result of covalent linkage of glucose to a lysine via formation of a Schiff base (22). This leads to loss of a positive charge of the molecule and a shift to the acidic region of the IEC chromatogram. Glycation typically occurs during the CHO cell culture process of antibody production when a high level of glucose is available in the cell culture medium. Glycation of the G0 variant results in an iso-form with the same mass as the G1 variant. In addition, the G1 variant is also glycated leading to the observed increased mass of 49,969 Da. Glyca-tion of the G2 variant leads to a new peak with a mass of 50,131 Da, which is only observed in the isolated acidic peak and not in the MS spectrum of the IEC main peak. The elution be-haviour of the IEC peak supports the hypothesized glycation.


dance Acidic IEC Peak in Fab 1 In addition to monoclonal antibod-ies, a therapeutic Fab (Fab 1) was also characterized by 2D-HPLC–MS. The material was temperature stressed for 8 weeks at 40 °C and degradation products were subsequently measured by IEC. The non-stressed material was measured as reference. For separation conditions refer to Table I. The IEC chromatogram of the stressed material showed one unknown peak, which was subsequently analyzed with the estab-lished 2D-HPLC–MS online reduction assay. The peak of interest is shown in Figure 5a.

The peak was analyzed and found to be a modified light chain with a mass delta of +16 Da in compari-son to the non-modified light chain. Based on three-dimensional X-ray structure measurements (data not shown), this mass shift most prob-ably represents an oxidation of an exposed tryptophan residue in the complementarity-determining region (CDR) of Fab 1.

The deconvoluted MS spectra are depicted in Figure 5b and 5c. Because

the fractionated peak is next to the main peak there is also a small signal of the native light chain, with the mass of 24,004 Da next to the modified ver-sion with a mass of 24,020 Da.

Further Experiments

Further experiments using SEC in the first dimension instead of IEC were performed. These tests were per-formed with antibody A and antibody B. The online trapping and reduction of fractionated peaks were compa-rable to the IEC experiments (data not shown here). This combination is most appropriate when mispaired species or fragment peaks have to be characterized.

An a lternative reducing agent, namely TCEP instead of DTT, was evaluated using exactly the same con-ditions such as temperature, concen-tration of the reducing agent, duration of reducing agent f low, and f low rate. TCEP was also tested with different antibodies and produced exactly the same results as the reduction with DTT (data not shown).

Conclusions

Characterization of product variants or post-translational modifications of therapeutic antibodies is essential because of their potential impact on product quality, safety, and efficacy (23); for example, a loss in potency can become a problem (24,25). To control those species various analyti-cal QC methods are applied that are predominantly HPLC-based. Until now the character izat ion of un-known peaks had to be performed by off line fractionation. This is a time-consuming process with the risk of introducing additional, unintended mod i f ic at ions . T he 2D -H PLC –QTOF-MS setup described here en-ables fully automated and rapid char-acterization of unknown peaks and is easy to use. It was possible to in-crease the sensitivity fivefold (0.2 μg of protein for each cut) compared to previous works (17). The system uses standard HPLC components and al-lows the use of the established QC methods for larger proteins (such as mAbs) in the first dimension without

any adaptations. Unknown peaks ob-served in routine QC measurements can be directly characterized with-out the need of re-conformation runs or bridging between methods. The use of multiple heart-cutting as part of the 2D-HPLC system offered easy analysis in the second dimension without the need to stop the chro-matographic f low. This approach has been successfully implemented for a plethora of biopharmaceutical development projects in our pipeline. The use of a reversed phase gradient for the separation of reduced mAb chains enables the identification and localization of modifications. Com-pared to conventional peak frac-tionation and characterization, the 2D-HPLC–MS setup described here yields a reduction in time-to-result of one order of magnitude, allowing sample characterization in a single day (Figure 6).

In some cases (for example, deami-dation) identification of the modi-fication can be improved by further reducing the size of antibody chains by digestion enzymes, like papain or trypsin. Further development work is ongoing to establish an additional di-gestion step for each fractionated peak in a 4D-HPLC–MS setup to achieve clear localization of modifications and verification of small mass shifts (1 Da for deamidation).

Acknowledgment

The work presented here was spon-sored by the Roche Global Strategy Team (GST) process. The authors wish to thank Rico Schaerer from Agilent for his support.

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the Max-Planck Institute for Molecular

Physiology, Dortmund, Germany, and

the Scripps Research Institute, San

Diego, California, USA. He went on to

do his Ph.D. at the University of Oxford,

UK, focusing on structural biology. Since

2011, Christian has worked for Roche

and is currently focused on analytical

development and quality control of

protein therapeutics in a clinical set-

ting. Anja Bathke is Group Head

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Hoffmann-La Roche, Biotech Analytics

in Basel, Switzerland. She is an expert

in MS protein characterization and

hyphenated techniques. Anja studied

biotechnology at the Fachhochschule

Lausitz - University of Applied Sciences,

Germany. Robert Kopf has worked

as a principal scientist in the Biotech

Analytics department of Hoffmann-La

Roche since 2004. He studied analytical

chemistry at the Reutlingen University,

Germany. He has been involved in HPLC

method development and validation for

small and large molecules for his whole

career. Christoph Gstöttner

is a PhD student in the Center for

Proteomics and Metabolomics at the

Leiden University Medical Center

(LUMC), Leiden, in The Netherlands.

Before that he studied biology at

Regensburg University, Germany.

Denis Klemm studied applied

chemistry at Reutlingen University,

Germany, with special focus on bioana-

lytics. Since 2017 Denis has worked at

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the development of the described char-

acterization system during their master

theses.

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Department of Chemistry at Wayne

State University in Detroit, Michigcan,

and the Cardiovascular Research

Institute at Wayne State University

School of Medicine. Santosh

Karki, Darrell D. Marshall,

Ellen. D. Inutan and Anil K.

Meher are with the Department of

Chemistry at Wayne State University.

Sara Madarshahian,

Madeline A. Fenner and

Charles N. McEwen are with

the Department of Chemistry &

Biochemistry at the University of the

Sciences in Philadelphia, Pennsylvania.

Direct correspondence to:


(Continued from page 17)


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32 Medical/Biological ADVERTISEMENT

Abuse of synthetic opioid prescription painkillers such

as fentanyl, along with a rapidly growing list of illicit

analogues, is a signifi cant public health problem. In

this study, we developed a simple dilute-and-shoot

method that provides a fast 3.5-min analysis of

fentanyl and related compounds (norfentanyl, acetyl

fentanyl, alfentanil, butyryl fentanyl, carfentanil,

remifentanil, and sufentanil) in human urine by

LC–MS/MS using a Raptor Biphenyl column.

In recent years, the illicit use of synthetic opioids has skyrocketed,

and communities worldwide are now dealing with an ongoing

epidemic. Of the thousands of synthetic opioid overdose deaths per

year, most are related to fentanyl and its analogues. With their very

high analgesic properties, synthetic opioid drugs such as fentanyl,

alfentanil, remifentanil, and sufentanil are potent painkillers that

have valid medical applications; however, they are also extremely

addictive and are targets for abuse. In addition to abuse of these

prescription drugs, the current opioid crisis is fueled by a growing

number of illicit analogues, such as acetyl fentanyl and butyryl fen-

tanyl, which have been designed specifi cally to evade prosecution

by drug enforcement agencies.

As the number of opioid drugs and deaths increases, so does

the need for a fast, accurate method for the simultaneous analysis

of fentanyl and its analogues. Th erefore, we developed this

LC–MS/MS method for measuring fentanyl, six analogues, and one

metabolite (norfentanyl) in human urine. A simple dilute-and-shoot

sample preparation procedure was coupled with a fast (3.5 min)

chromatographic analysis using a Raptor Biphenyl column. Th is

method provides accurate, precise identifi cation and

quantitation of fentanyl and related compounds, making

it suitable for a variety of testing applications, including

clinical toxicology, forensic analysis, workplace drug

testing, and pharmaceutical research.

Experimental Conditions

Sample Preparation

Th e analytes were fortifi ed into pooled human urine.

An 80 μL urine aliquot was mixed with 320 μL of

70:30 water–methanol solution (fi ve-fold dilution)

and 10 μL of internal standard (40 ng/mL in metha-

nol) in a Th omson SINGLE StEP fi lter vial (Restek

cat. #25895). After fi ltering through the 0.2 μm PVDF

membrane, 5 μL was injected into the LC–MS/MS.

Analysis of Fentanyl and Its Analogues in Human Urine by LC–MS/MSShun-Hsin Liang and Frances Carroll, Restek Corporation

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

Time (min)

Figure 1: The Raptor Biphenyl column effectively separated all target compounds in urine with no observed matrix interferenc-es. Peak elution order: norfentanyl-D5, norfentanyl, remifent-anil, acetyl fentanyl-13C6, acetyl fentanyl, alfentanil, fentanyl-D5, fentanyl, carfentanil-D5, carfentanil, butyryl fentanyl, sufentanil-D5, sufentanil.

Table I: Analyte transitions

AnalytePrecursor

Ion

Product Ion

Quantifi er

Product Ion

Qualifi erInternal Standard

Norfentanyl 233.27 84.15 56.06 Norfentanyl-D5

Acetyl fentanyl 323.37 188.25 105.15 Acetyl fentanyl-13C6

Fentanyl 337.37 188.26 105.08 Fentanyl-D5

Butyryl fentanyl 351.43 188.20 105.15 Carfentanil-D5

Remifentanil 377.37 113.15 317.30 Norfentanyl-D5

Sufentanil 387.40 238.19 111.06 Sufentanil-D5

Carfentanil 395.40 113.14 335.35 Carfentanil-D5

Alfentanil 417.47 268.31 197.23 Acetyl fentanyl-13C6

Norfentanyl-D5

238.30 84.15 — —

Acetyl fentanyl-13C6

329.37 188.25 — —

Fentanyl-D5

342.47 188.27 — —

Sufentanil-D5

392.40 238.25 — —

Carfentanil-D5

400.40 340.41 — —

Medical/Biological 33ADVERTISEMENT

Restek Corporation110 Benner Circle, Bellefonte, PA 16823

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Calibration Standards and Quality Control Samples

Th e calibration standards were prepared in pooled human urine at

0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, 10.0, 25.0, and 50.0 ng/

mL. Th ree levels of QC samples (0.75, 4.0, and 20 ng/mL) were

prepared in urine for testing accuracy and precision with estab-

lished calibration standard curves. Recovery analyses were per-

formed on three diff erent days. All standards and QC samples

were subjected to the sample preparation procedure described.

LC–MS/MS analysis of fentanyl and its analogues was

performed on an ACQUITY UPLC instrument coupled with

a Waters Xevo TQ-S mass spectrometer. Instrument conditions

were as follows, and analyte transitions are provided in Table I.

Analytical column: Raptor Biphenyl (5 μm,

50 mm × 2.1 mm; cat. #9309552)

Guard column: Raptor Biphenyl EXP guard column

cartridge, (5 μm, 5 mm × 2.1 mm;

cat. #930950252)

Mobile phase A: 0.1% Formic acid in water

Mobile phase B: 0.1% Formic acid in methanol

Gradient Time (min) %B

0.00 30

2.50 70

2.51 30

3.50 30

Flow rate: 0.4 mL/min

Injection

volume: 5 μL

Column temp.: 40 °C

Ion mode: Positive ESI

Results

Chromatographic Performance

All eight analytes were well separated within a 2.5-min gradient elu-

tion (3.5-min total analysis time) on a Raptor Biphenyl column (Fig-

ure 1). No signifi cant matrix interference was observed to negatively

aff ect quantifi cation of the fi ve-fold diluted urine samples. Th e 5-μm

particle Raptor Biphenyl column used here is a superfi cially porous

particle (SPP) column. It was selected for this method in part because

it provides similar performance to a smaller particle size fully porous

particle (FPP) column, but it generates less system backpressure.

Linearity

Linear responses were obtained for all compounds and the

calibration ranges encompassed typical concentration levels

monitored for both research and abuse. Using 1/x weighted linear

regression (1/x2 for butyryl fentanyl), calibration linearity ranged

from 0.05 to 50 ng/mL for fentanyl, alfentanil, acetyl fentanyl,

butyryl fentanyl, and sufentanil; from 0.10 to 50 ng/mL for

remifentanil; and from 0.25 to 50 ng/mL for norfentanyl and

carfentanil. All analytes showed acceptable linearity with r2 values

of 0.996 or greater and deviations of <12% (<20% for the lowest

concentrated standard).

Accuracy and Precision

Based on three independent experiments conducted on multiple

days, method accuracy for the analysis of fentanyl and its ana-

logues was demonstrated by the %recovery values, which were

within 10% of the nominal concentration for all compounds at

all QC levels. Th e %RSD range was 0.5–8.3% and 3.4–8.4%

for intraday and interday comparisons, respectively, indicating ac-

ceptable method precision (Table II).

Conclusions

A simple dilute-and-shoot method was developed for the quan-

titative analysis of fentanyl and its analogues in human urine.

Th e analytical method was demonstrated to be fast, rugged, and

sensitive with acceptable accuracy and precision for urine sample

analysis. Th e Raptor Biphenyl column is well suited for the analy-

sis of these synthetic opioid compounds and this method can be

applied to clinical toxicology, forensic analysis, workplace drug

testing, and pharmaceutical research.

Table II: Accuracy and precision results for fentanyl and related compounds in urine QC samples

QC Level 1 (0.750 ng/mL) QC Level 2 (4.00 ng/mL) QC Level 3 (20.0 ng/mL)

AnalyteAverage Conc.

(ng/mL)

Average %

Accuracy %RSD

Average Conc.

(ng/mL)

Average %

Accuracy %RSD

Average Conc.

(ng/mL)

Average %

Accuracy %RSD

Acetyl fentanyl 0.761 102 1.54 3.99 99.7 2.08 19.9 99.3 0.856

Alfentanil 0.733 97.6 3.34 3.96 98.9 8.38 20.9 104 6.73

Butyryl fentanyl 0.741 98.9 6.29 3.77 94.3 6.01 20.8 104 4.95

Carfentanil 0.757 101 7.34 3.76 94.0 4.64 20.6 103 4.24

Fentanyl 0.761 102 1.98 3.96 99.1 2.31 19.9 99.6 1.04

Norfentanyl 0.768 103 6.50 4.04 101 1.84 20.1 101 2.55

Remifentanil 0.765 102 3.42 3.97 99.2 3.68 20.8 104 4.14

Sufentanil 0.752 100 1.67 3.93 98.3 1.28 20.1 100 0.943

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References. Number the literature ci-tations in the text consecutively in order of appearance and indicate them by Arabic numerals in parentheses. Num-ber each reference separately. Group the references at the end of the manuscript in the order of their appearance in the text, not alphabetically.

Use Chemical Abstracts Service Source Index for journal abbreviations. Use the following format for references:(1) R. Salzer and H.W. Siesler, Infrared and

Raman Spectroscopic Imaging (Wiley-VCH, Weinheim, 2009), pp. 90–103.

(2) P. Matousek, Appl. Spectrosc. 60, 1341 (2006).

Author Benefits

Publishing your work in Spectroscopy has many advantages. The size of Spectros-copy’s audience, with more than 27,000 readers, makes Spectroscopy the ideal vehicle for communicating information of broad significance to the spectroscopy community. Contributed technical papers published in Spectroscopy have passed a thorough peer-review process. Our pool of referees includes the members of our dis-tinguished Editorial Advisory Board.

Lasers and Optics Interface Column

We also accept contributed manuscripts for the “Laser and Optics Interface” col-umn. Tutorials, reviews, and experimental articles can all be suitable for this column.

Special Supplements

A number of supplemental issues are planned for 2018, for which we also in-vite contributions. Manuscripts for the supplements on Raman, IR, ICP & ICP-MS, portable/handheld spectroscopy, and food and beverage analysis should be 2000–2500 words long, plus up to six figures and tables, and should include an abstract of approximately 150–200 words. We are still accepting submissions for the following issues:

Raman supplement, June 2018

Extended Submission deadlines:Abstracts: March 23, 2018Manuscripts: April 20, 2018

IR supplement, August 2018

Submission deadlines:Abstracts: March 8, 2018Manuscripts: May 15, 2018

ICP & ICP–MS supplement,

September 2018

Submission deadlines:

Abstracts: April 6, 2018Manuscripts: June 15, 2018

Food & Beverage Analysis

supplement, November 2018

Submission deadlines:Abstracts: May 4, 2018Manuscripts: July 9, 2018

Portable/Handheld Spectroscopy

supplement, November 2018

Submission deadlines:Abstracts: May 4, 2018Manuscripts: July 9, 2018

Current Trends in Mass Spectrometry supplement series

Our supplement series on mass spectrom-etry appears four times a year: in March, May, July, and October. Manuscripts for this series should be approximately 2500–3500 words long, including an abstract of approximately 150–200 words, plus up to eight figures and tables. We are still accept-ing submissions for the following issues: Submission Deadlines:May 2018 issue (extended deadines)Abstracts: March 16, 2018Manuscripts: April 2, 2018

July 2018 issueAbstracts: March 16, 2018Manuscripts: May 14, 2018

October 2018 issueAbstracts: April 2, 2018Manuscripts: June 8, 2018

Contact Us

For more information about contrib-uting to Spectroscopy, please contact

Laura Bush, Editorial Director

Spectroscopy

+1.732.346.3020

[email protected]

www.spectroscopyonline.com ◾

Call for PapersSpectroscopy invites researchers to submit their work for publication.

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