Acrylam

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Acrylamide pg 2-4

Aflatoxins pg 5

Melamine pg 6

Sudan dyes pg 7

Sulfonamides pg 8-9

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Rapid and Reproducible Extraction of Acrylamide in French Fries using a Single SPE Sorbent - Strata™-X-C

Acrylamide (MW 71, m.p. 84.5 °C, logP 0.67) belongs to the enamide class of organic compounds and is an extremely important indus-trial chemical. The polyacrylamide family of polymers and copoly-mers derived from this monomer find applicability in widely differing areas such as clarification of drinking water, flocculants for waste water treatment, oil recovery, soil conditioning, agriculture and biomedical purifications. However, the neurotoxicity of acrylamide poses a serious health risk and leads to a reluctance by the public at large to accept the polyacrylamides as safe materials.1 To add to these concerns, the presence of acryamide has recently been detected in food products prepared at high temperatures.2,3 Earlier methods for the detection of acrylamide involved liquid-liquid extraction followed by derivatization (bromination) and GC/ECD or GC/MS analysis.4,5 The high detection limits associated with such methods limit their applicability for the determination of low levels of acrylamide in food matrices, in addition to being tedious and laborious.

Recently published methods for the determination of acrylam-ide6,7,8 utilize either one or two successive solid phase extraction (SPE) sorbents for sample preparation before LC/MS detection. The method that uses a single reversed-phase sorbent is based on nonretentive SPE, removing only the hydrophobic components of the sample while the acrylamide passes, unretained, through the sorbent. A more extensive clean up procedure has been proposed that requires two SPE sorbents. In this method, a hydrophilic/ lipophilic sorbent is applied first for the removal of most sample components. The sample is then passed through a second tube that contains a mixed mode sorbent that refines the extract of the target analyte - acrylamide. These methods provide cleaner extractions for analysis, but are time consuming and costly.

In this application note, we present a simple and efficient method which uses a single SPE sorbent - Strata-X-C - for the extrac-tion of acrylamide from a food sample (French fries). Strata-X-C is a revolutionary, patent pending polymeric resin that has been functionalized with polar and strong cation exchange groups. As a result, Strata-X-C exhibits numerous retention mechanisms in-cluding hydrophobic, hydrogen bonding, π-π and strong cation exchange, making it ideal for the extraction of acrylamide from food samples. All of these retention mechanisms will come into play for the effective cleanup of the acrylamide sample.

InstrumentationHPLC: Agilent 1100 series (Palo Alto, CA USA)

MS: Bruker Esquire 2000 Ion-Trap MS analyzer, (Billerica, MA USA) Ion source APCI in positive ion mode MRM (acrylamide m/z:72→55, acrylamide-d3 m/z:75→58).

LC columns: Synergi™ Hydro-RP 4 µm 250 x 3.0 mm (LC/MS) Synergi™ Polar-RP 4 µm 150 x 4.6 mm (HPLC/UV)

Experimental Conditions Specimen preparation: Add 50 mL water to 10 g pulverized frozen French fries; mix or homogenize for 20 minutes. Centrifuge the decanted solution at 10000 rpm for 15 minutes. Spike sample with internal standard - acrylamide-d3. Apply supernatant to a condi-tioned Strata-X-C tube as described below.

SPE method1. Condition: apply 2 x 1 mL methanol, followed by 2 x 1 mL water to Strata-X-C tube at a flow rate of 2 mL/min, controlled with a vacuum manifold.

2. Sample Load: apply 1 mL supernatant (prepared as described above) at a flow rate of less than 0.5 mL/min.

3. Dry: 30 seconds under vacuum (10-12 inches Hg).

4. Elution: 1 mL water at a flow rate less than 0.5 mL/min; collect eluate in a sample vial; draw any residual water from the sorbent by applying full vacuum. This eluate is ready for LC/MS analysis.

Important! The recommended volumes are for 100 mg of sorbent bed mass. The volumes will have to be adjusted for smaller or larger sorbent masses.

Analysis: A calibration curve was generated by spiking acrylamide and acrylamide-d3 (both prepared in water) at different concentra-tions in 1 mL of French fries supernatant. Two unspiked samples of supernatant served as blanks.

Results and DiscussionFor retaining a polar and highly water-soluble (215.5 g/100 mL water) analyte such as acrylamide on an SPE sorbent, one has to harness every possible mode of polar interaction. It is well known that enamides can be protonated with Bronsted acids to form an N-acyliminium cationic species in solution.9,10 Such behavior can be favorably utilized to retain acrylamide on a cation exchange sorbent such as Strata-X-C, which can also undergo other polar interactions as well.

Scheme 1. Protonation of acrylamide by Strata-X-C.

Liming Peng, Tivadar Farkas, Lawrence Loo, Art Dixon, James Teuscher, Krishna Kallury Phenomenex, Inc. Torrance, CA USA

CH2 =CH-C=O

NH2

Acrylamide

CH2 -CH=C-O

NH2

-+

H (from sulfonic acid of sorbent)+

CH2 -CH=C-O-H

NH2

+

O-Protonated acrylamide

CH2 =CH-C=O

NH3+

N-Protonated acrylamide

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The strongly acidic sulfonic acid moieties on Strata™-X-C can furnish the hydronium ion for protonating acrylamide, as shown in Scheme 1. There is the choice of using either silica- or poly-mer-based cation exchange sorbent for extracting acrylamide. However, there are several advantages in using the polymeric sorbent. First, Strata-X-C has larger surface area (800 m2/g vs. 500 m2/g) and hence greater retention than silica based materi-als. Second, there is a greater concentration of ion exchange moieties on a polymeric surface than a silica surface. Further-more, a polymeric sorbent does not carry any silanol groups as the silica surface does. This is significant as silanol groups interact with sulfonic acid groups through more than one mecha-nism and hence render some of the sulfonic acid groups unavail-able for interactions with acrylamide. A comparative evaluation of Strata® SCX and Strata-X-C sorbents reveals the silica-based SCX material yields around 50 % recovery of acrylamide, while the recovery from the polymeric Strata-X-C sorbent is near quantita-tive. Lastly, the polymeric sorbent has more retention modes for interaction with sample interferences. For extracting acrylamide from French fries, one has to use water not only due to the high solubility of acrylamide in this solvent, but also to minimize disso-lution of hydrophobic molecules in this food product. Furthermore, in developing an SPE protocol for acrylamide, the wash step has to be excluded in view of its solubility in water. Elution with water also has the advantage of eliminating the desorption of all hydro-phobic impurities which remain adsorbed on the sorbent.

Figure 1. LC/UV chromatogram of acrylamide in a French fries extract using Strata-X-C.

2.9

22

- A

cry

lam

ide

2 µg/mL acrylamide in0.2 g French fries; UVdetection at 210 nm

Norm

40

30

20

10

0

0 2 4 6 8 min

Column: Synergi™ Polar-RP 4 µm Dimensions: 150 x 3.0 mm

Mobile Phase: 94:6 (v:v) water: CH3CN at 0.4 mL/minInjection Volume: 10 µL

Figure 2. LC/MS/MS extracted ion chromatograms for m/z: 72 ->55. Intens

x15

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0.75

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0 1 2 3 4 5 6 7 8 min

( )CH CH CNH

+O

CH CH C O+

23

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2

0.2 g French fries10 ng/mL acrylamide in 0.2 g french fries500 ng/mL acrylamide in 0.2 g French fries

Column: Synergi Polar-RP 4 µm Dimensions: 250 x 3.0 mm

Mobile Phase: 4:6 (v:v) water: methanol (both contain 0.1 % formic acid) at 0.5 mL/min

Injection Volume: 25 µL

As shown in Figure 1, HPLC(UV) chromatograms recorded at 210 nm demonstrate that extraction using Strata-X-C gives clean extracts with low background and a well separated acrylamide peak from all matrix components. Figure 2 shows the ion chro-matogram for the transition of the protonated molecular ion of acrylamide (m/z 72) to the acryloyl cation (m/z 55) through the elimination of NH3 (ammonia). The mobile phase for LC/MS comprised of 96:4 water/methanol containing 0.1 % formic acid. The data in this figure shows the high sensitivity of the MS mode of detection, even for trace levels of acrylamide. The reproduc-ibility of extraction demonstrates excellent precision at three different concentration levels (Table 1). The absolute recoveries were determined by comparing peak areas for same level stan-dards and for spiked acrylamide extracts subtracted for blank. The linearity was studied in two different concentration ranges: 10-500 ng/mL acrylamide (with IS at 100 ng/mL), (see Figure 3) and 100-2000 ng/mL (with IS at 500 ng/mL) as shown in Table 1. The linearity based on external standard was also evaluated. Results demonstrate good linearity in all cases with values of R2 > 0.998.

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Rapid and Reproducible Extraction of Acrylamide in French Fries using a Single SPE Sorbent - Strata™-X-C (cont’d)

ConclusionsThe novel selectivity of Strata-X-C allows for the rapid extraction of acrylamide from French fries using a simple and reproducible method. Since the method uses a single SPE sorbent it is an attractive alternative to currently applied or proposed methods for the extraction of acrylamide in food sam-ples. The versatility of this clean up method allows for the analysis of acrylamide present in food at ppb or ppm concentration levels, giving chromatograms with relatively little background interfer-ence. It also eliminates the necessity of column flushing, as sample components are not present to build up under weak mobile phase conditions (high percentage of water).

Table 1. Results of SPE & LC/MS/MS method validation

Concentration (ng/mL) RSD, % (n=6) Recovery, % (n=6)

50 9.55 >78

500 2.20 >84

2000 0.82 >91

R2

10-500 (5 pts) >0.999

100-2000 (5 pts) >0.999

100-2000*(5 pts) >0.999

* external standard

Ordering InformationPart No. Description Unit8B-S029-TAK Strata-X-C 30 mg/1 mL Tubes 100/Box8B-S029-UBJ Strata-X-C 60 mg/3 mL Tubes 50/Box8B-S029-FBJ Strata-X-C 200 mg/3 mL Tubes 50/Box8B-S029-HBJ Strata-X-C 500 mg/3 mL Tubes 50/Box8B-S029-ECH Strata-X-C 100 mg/6 mL Tubes 30/Box8B-S029-FCH Strata-X-C 200 mg/6 mL Tubes 30/Box8B-S029-HCH Strata-X-C 500 mg/6 mL Tubes 30/Box8B-S029-EDG Strata-X-C 100 mg/12 mL Giga Tubes 20/Box8B-S029-HDG Strata-X-C 500 mg/12 mL Giga Tubes 20/Box8B-S029-JDG Strata-X-C 1 g/12 mL Giga Tubes 20/Box8E-S029-AGB Strata-X-C 96-Well Plate 10 mg/well 2/Box8E-S029-TGB Strata-X-C 96-Well Plate 30 mg/well 2/Box8B-S029-UGB Strata-X-C 96-Well Plate 60 mg/well 2/Box00F-4336-E0 Synergi Polar-RP 4 µm 150 x 4.6 mm ea00G-4375-Y0 Synergi Hydro-RP 4 µm 250 x 3.0 mm eaAH0-6023 12-Position SPE Vacuum Manifold ea

References1. Caulfield, et al., Chem. Rev., 2002, 102, 3067.

2. Tareke et al., Chem. Res. Toxicol., 2002, 13, 517.

3. International Agency for Research on Cancer, 1994, VI:60, 389.

4. Castle et al., J. Sci Food Agric., 1991, 54, 549.

5. EPA(USA) Method 8032A.

6. Mann, et al. “Acrylamide Analysis Using LC-MS/MS” Waters Corporation.

7. FDA(USA) Center for Food Safety and Applied Nutrition Office of Plant & Dairy Foods & Beverages, “Detection and Quantitation of Acrylamide in Foods” July 23, 2002.

8. Kawata, et al., J. Chromtogr. A, 2001, 911, 75.

9. Wang et al., J. Org. Chem., 2001, 66, 8215.

10. Speckamp, et al., Tetrahedron, 2000, 56, 3817.

Figure 3. Linearity of extraction of acrylamide using the Strata-X-C method.

Calibration curve prepared by spiking 10, 20, 50 (n=6), 100, 500 (n=6) ng/mL acrylamide, respectively, and 100 ng/mL IS acrylamide-d3 into 1 mL of 0.2 g/mL French fries extract. Detection: LC/MS/MS.

Linearity of Strata X-C for Acrylamide

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 100 200 300 400 500 600

Concentration (ng/mL)

Acr

ylam

ide

/ IS

y = 0.0116x + 0.2089

R2 = 0.9979

Aflatoxins

5

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AP

P ID

163

56

Column: Gemini 5 µm C18 110 ÅDimensions: 150 x 3.0 mm ID

Part No.: 00F-4435-Y0Mobile Phase: A: 10 mM Ammonium Acetate, pH 4.5

B: Acetonitrile (70:30)Elution Type: Isocratic

Flow Rate: 0.5 mL/minCol. Temperature: Ambient

Detection: Mass Spectrometer (MS) AmbientSamples: 1. Aflatoxin G2

2. Aflatoxin B23. Aflatoxin G14. Aflatoxin B1

Column: Luna 3 µm PFP(2) 100 ÅDimensions: 150 x 4.6 mm ID

Part No.: 00F-4447-E0Mobile Phase: A: 10 mM Ammonium Acetate, pH 4.5

B: Acetonitrile (70:30)Elution Type: Isocratic

Flow Rate: 1.0 mL/minCol. Temperature: Ambient

Detection: UV @ 220 nmSamples: 1. Aflatoxin G2

2. Aflatoxin B23. Aflatoxin G14. Aflatoxin B1

Luna® 3 µm PFP(2)

Aflatoxin B1, B2, G1, and G2 Determination by HPLC

Aflatoxins are a secondary metabolic product produced by vari-ous strains of the Aspergillus flavus and Aspergillus parasiticus fungi. Once moisture and temperature levels increase significantly this particular fungi is known to infect produce. Some commonly infected crops include oil seeds, cereals, spices, dried fruits, and tree nuts.

In humans and other animals, aflatoxins are known to be carcino-genic. If contaminated foodstuff is ingested, these mycotoxins will target the liver and can cause a variety of health issues. Out of the four main aflatoxins, B1 is notably the most toxic. In addition to general health concerns, Aspergillus and its aflatoxins also have a large effect economically. Fungal infection can lead to losses in crop yield and quality, while aflatoxin presence can lead to live-stock/poultry death or reduced growth rates.

HPLC in combination with UV or MS detection remains one of the most sensitive and efficient ways of determining aflatoxins in various matrices. The following covers detailed methods for de-termination of aflatoxin B1, B2, G1, G2 using HPLC with UV and MS detection.

Gemini® 5 µm C18

Terrell Matthews, Product Manager Phenomenex, Inc. Torrance, CA USA

1 2 3 4 5 6 7 8 9 10 11 12 min0

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HPLC method using Luna® HILIC

Column: Luna 3 μm HILIC, 100 x 2.0 mmPart No.: 00D-4449-B0

Flow Rate: 0.4 mL/minMobile Phase: (90:10) Acetonitrile:100 mM Ammonium

formate, pH 3.2Analysis: Isocratic

Run Time: 3.5 minutesInjection Volume: 5 μL

Temperature: AmbientMRM: m/z 127 – 85, 127 – 60 ESI Positive mode

50 ng/ml Melamine standard, (95:5) ACN:H2O 10 ng/ml Melamine standard (95:5) ACN:H2O

02000400060007613 2.02

0

5000

9120 2.00

0

5000

9127 2.00

0.2 0.6 1.0 1.4 1.8 2.2 2.6 2.8 min

0.2 0.6 1.0 1.4 1.8 2.2 2.6 2.8 min

0.2 0.6 1.0 1.4 1.8 2.2 2.6 2.8 min

0.2 0.6 1.0 1.4 1.8 2.2 2.6 2.8 min

0

5000

8787 2.02

2.05

2.03

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1000

1500

20002273

0200

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1400

18002140

0.2 0.6 1.0 1.4 1.8 2.2 2.6 2.8 min

SPE and HILIC HPLC Strategy for Determination of Melamine

As the world faces its second melamine scare in two years, there has been a global concern over international food product imports and food safety testing. The compound itself has a relatively low toxicity to humans. However, when melamine combines with cya-nuric acid, it forms an insoluble salt crystal that causes kidney failure in animals and humans.

Nitrogen is a major component of amino acids, which are the building blocks for proteins. With 6 nitrogen atoms in its structure, melamine has been added to some foods to falsely increase their protein content. This manipulation is possible because the stan-dard method to determine protein content in food is the Kjeldahl method, which measures the amount of organic nitrogen in the sample. This method is non-specific, so any other nitrogen pres-ent in the sample will cause the test to show higher protein levels than are actually present. By adding melamine to their products, food producers have found a way to cover up diluting their prod-ucts with the consumer suffering the consequences.

The determination of melamine in food products is of great impor-tance, especially with the recent discovery of melamine in baby formula. The following methodology can be used to determine the levels of melamine in food products. In this method, SPE clean up of the sample is followed with analysis by LC/MS/MS.

SPE method - specific targeting using Strata™-X-C

Column: Strata-X-C, 100 mg/6 mLPart No.: 8B-S029-ECH

Condition: 2 mL MethanolEquilibrate: 2 mL 0.1% TFA in water, pH<4

Load: Load sample and allow to slowly drip through cartridge at a rate of 1-2dps (drops per second)

Wash A: 2 mL 0.1% TFA in Water, pH<4Wash B: 2 mL MethanolElution: 2 mL Methanol / 5 % NH4OH. Use a 2 stage

elution (2 x 1 mL)

Terrell Matthews, Product Manager Phenomenex Inc., Torrance, CA, USA

Sud

an Dyes

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Figure 1. Separation of Sudan Dyes

Ordering Information Part No. Description Unit00F-4336-E0 Synergi™ 4μm Polar-RP 150 x 4.6 mm ea

ResultsThe choice of a polar-embedded phenyl phase (Synergi™ Polar-RP) and the use of methanol in the mobile phase resulted in a good separation of the two isomeric Sudan dyes (Sudan Red B and Sudan IV). The method enables the quantitation of Sudan I-IV and Sudan Red B in one run within 15 minutes.

References1. Commission Decision 20 June 2003. Official Journal of the European Union

L154/114.

2. Annual Report 2004. Rapid Alert System for Food and Feed. European Commission Health & Consumer Protection Directorate General.

Column: Synergi 4 μm Polar-RP 80 ÅDimensions: 150 x 4.6 mm ID

Part No.: 00F-4336-E0Mobile Phase: Water/Acetonitrile/Methanol (15:20:65)

Elution Type: IsocraticFlow Rate: 1.0 mL/min

Col. Temperature: 30 ˚CDetection: UV @ 480 nmSamples: 1. Sudan I

2. Sudan II3. Sudan III4. Sudan Red B5. Sudan IV

min0 2 4 6 8 10 12 14

mAU

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20

40

60

80

100

1201

2 3

4

5

Easy Isocratic HPLC Determination of Sudan Dyes

In May 2003, France issued a warning against chili powders from India that have been contaminated with the Sudan dye Sudan I. Sudan dyes are azo dyes.1 These can be metabolized to pri-mary amines, which are suspected carcinogenic compounds. Therefore, in the European Union it is not allowed to use these dyes for coloring food or foodstuff. Since June 2003, importers or food manufacturers must include a certificate of analysis showing the absence of these compounds (Sudan I - IV)2 in all shipments of chili powder and products containing chili powder. The EU national food administrations are forced to control this on an on-going basis. As evidence of this ongoing concern, in February 2005 the largest recall of processed food (fish sauce, worchester sauce, noodle soup, and pizza) occured in the United Kingdom due to chile powder contaminated with Sudan I.

IntroductionThe goal of this work was the development of an easy and iso-cratic HPLC method for the determination of Sudan dyes (Sudan I-IV and Sudan Red B). The separation of the two isomers Sudan IV and Sudan Red B is especially challenging for the chromatog-rapher as it is not possible to distinguish between these two com-pounds in LC/MS if they are co-eluting. Conventional C18 phases do not have the right selectivity to solve this task in an easy man-ner. Thus we decided to use a unique, polar-embedded phenyl phase (Synergi™ Polar-RP) for the method development. To en-hance pi-pi interactions, methanol was choosen as the organic modifier.

Instrumentation/EquipmentAnalysis was performed using an HP 1100 LC system, equipped with a quaternary pump, in-line degasser, diode-array detector and autosampler (Agilent Technologies, Palo Alto, CA, USA). The HPLC column used for the analyses was a Synergi™ Polar-RP 150 x 4.6 mm (Phenomenex, Torrance, CA, USA). All solvents were HPLC grade and were obtained from Burdick and Jackson (Mus-kegon MI, USA). All Sudan dyes were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA).

ExperimentalChromatographic conditions: A tertiary mobile phase of water/ac-etonitrile/methanol (15:20:65) was used, with a flow rate of 1 mL/min. The column temperature was 30 °C. UV-Visible detection was recorded at a wavelength of 480 nm.

Scott Waite, Dirk Hansen, and Michael McGinley Phenomenex Inc., Torrance, CA, USA

AP

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158

90

Sul

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Extraction and Analysis of Sulfonamides from Honey by LC/MS/MS using Strata™X-C Polymeric SPE Sorbent and Gemini® C18 HPLC Column

IntroductionAmerican and European Foulbrood (AFB and EFB) in honeybees is caused by two bacterial species, Paenibacillus larvae and Melis-sococcus pluton, respectively. For control of these bacteria, only a few antibiotics are recommended in Europe and the U.S.A. How-ever, a variety of antibacterial agents are used, the sulfa drugs being the most popular. Residues of these antibiotics are quite often found in honey samples and are of concern to consumers around the world due to toxic or allergic reactions. Earlier sample preparation methods involved using silica-based C181 or neutral polymers as sorbents2 with an aqueous wash prior to elution of sulfonamides. In this communication, we describe a simple and effective method for clean-up and quantitation of antibacterial sulfonamides from honey using the polymeric strong cation ex-change SPE sorbent Strata-X-C and a Gemini HPLC column.

ExperimentalMaterials: Honey used was obtained from a local supermarket. All solvents and chemicals are from Aldrich, Milwaukee, WI. The Stra-ta-X-C cartridges (60 mg/3 mL) for solid phase extraction (SPE) and Gemini C18 columns (150 x 3.0 mm, 5 μm) for LC/MS are from Phenomenex, Torrance, CA. An Agilent 1100 HPLC system coupled to an API 3000 mass spectrometer (ESI source) was used for analysis of elution fractions from SPE.

Solid Phase Extraction: The cartridge is conditioned with 2 mL methanol, followed by 2 mL of deionized water. The honey sample (1 gram) is acidified with 1 mL of 2 M hydrochloric acid, sonicated for 30 min and then treated with 0.3 M citric acid in water so as to make up the total volume to 5 mL. The sulfa drugs are spiked into this diluted honey solution and loaded onto the cartridge. Washing was done with 4 mL of water (in two aliquots), then with 4 mL of 50:50 methanol/acetonitrile (in two aliquots) and then the cartridge was dried for 2 min at 10” of Hg pressure. Elution was carried out with 2 mL of 2 % ammonium hydroxide in methanol. Internal standard was then added and the eluate evaporated under a slow stream of nitrogen at 40 °C. The residue was reconstituted into 100 μL of mobile phase.

Table 1 Physicochemical Characteristics and Recovery Yields of Sulfa Drugs

Analytes Spiked Conc.

(ng/mL)

MRM Log P of

Analyte

pKa of Analyte

% Re-covered

% RSD

Sulfanilamide 100 173 -> 93.1

-0.62 2.4, 10.4

34 % 2-5%

Sulfathiazole 100 256 -> 92.0

0.09 2.08, 7.07

81 % 2-5%

Sulfamerazine 100 265.1 -> 108.2

0.54 1.16, 1.54, 2.0, 9.55

78 % 2-5%

Sulfamethoxazole 100 254.1 -> 92.0

1.58 1.83, 5.65

94 % 2-5%

Sulfaquinoxaline 100 301.2 -> 92.1

1.68 1.86, 5.56

IS 2-5%

Figure 1. LC/MS/MS of SPE extract of sulfa drugs (see Table 1 for MRM details)

Shahana Huq and Krishna Kallury Phenomenex Inc., Torrance, CA, USA

Liquid Chromatography

Column: Gemini 5 μm C18Part No.: 00F-4435-Y0

Dimensions: 150 x 3.0 mmMobile Phase: A: 0.1 % Formic Acid in Water

B: 0.1 % Formic Acid in AcetonitrileGradient: A/B 90:10 to 30:70 in 8 min

Flow Rate: 600 μL/minDetection: API 3000 LC/MS/MS with ESI+ (Turbolon

Spray), heater gas flow 7000 cc/min, heater temp. 425 ˚C

6.0e5

4.0e5

2.0e5

00 2 4 6 8 10 12 14 min

6002.26 Sulfanilamide

173.1/93.1

400

200

00 2 4 6 8 10 12 14 min

2.5e52.8e5 6.01 Sulfamethoxazole

265.1/92.0

1.0e5

5.0e4

00 2 4 6 8 10 12 14 min

1.5e5

2.0e5

0 2 4 6 8 10 12 14 min

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00 2 4 6 8 10 12 14 min

0 2 4 6 8 10 12 14 min

6.0e54.06 Sulfathiazole

256.0/92.1

4.31 Sulfamerazine265.1/108.2

6.66 Sulfaquinoxaline301.2/92.1

4.0e5

2.0e5

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3.0e5

2.0e5

1.0e5

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Result and DiscussionHoney is a conglomeration of several classes of compounds that include carbohydrates, aliphatic carboxylic acids, amino acids, proteins and minerals. Such a matrix presents a challenge for isolating the antibiotic contaminants, especially sulfa drugs. Sulfa drugs carry aromatic amino groups, which can react with the sug-ars forming Schiff’s bases. Hence acidic conditions are needed to break up such interactions. The cation exchange sorbent Strata™-X-C is ideal for eliminating the matrix components since sulfa drugs form strong ionic bonds with the sulfonic acid moieties on this sorbent. This facilitates stronger wash with organic solvents to get rid of the organic impurities while retaining the sulfa drugs. Figure 1 and Table 1 show the LC/MS/MS graphics and recovery data, respectively. Sulfanilamide is the only compound that shows lower recovery, due to its extremely polar characteristics (see Ta-ble 1 for log P values of the sulfa drugs studied). The strong organ-ic wash (see experimental section) removes part of sulfanilamide, such a wash being necessitated by the complexity of the honey matrix. With Strata-X-C, sulfa drugs including sulfanilamide are all recovered from plasma matrices in excellent yields3, demonstrat-ing that the sorbent is capable of retaining sulfanilamide during pure methanol wash. Also, part of the lower recovery yield can be attributed to the aromatic primary amino moiety of sulfanilamide (the most basic amongst sulfa drugs) forming a Schiff’s base with the sugars, an aspect already stressed in the literature2.

Figure 1. Cont’d

6.0e5

4.0e5

2.0e5

00 2 4 6 8 10 12 14 min

6002.26 Sulfanilamide

173.1/93.1

400

200

00 2 4 6 8 10 12 14 min

2.5e52.8e5 6.01 Sulfamethoxazole

265.1/92.0

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0 2 4 6 8 10 12 14 min

600

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00 2 4 6 8 10 12 14 min

0 2 4 6 8 10 12 14 min

6.0e54.06 Sulfathiazole

256.0/92.1

4.31 Sulfamerazine265.1/108.2

6.66 Sulfaquinoxaline301.2/92.1

4.0e5

2.0e5

0

3.0e5

2.0e5

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Ordering Information Part No. Description Unit8B-S029-TAK Strata-X-C 30 mg/1 mL Tubes 100/Box8B-S029-UBJ Strata-X-C 60 mg/3 mL Tubes 50/Box8B-S029-FBJ Strata-X-C 200 mg/3 mL Tubes 50/Box8B-S029-HBJ Strata-X-C 500 mg/3 mL Tubes 50/Box8B-S029-ECH Strata-X-C 100 mg/6 mL Tubes 30/Box8B-S029-FCH Strata-X-C 200 mg/6 mL Tubes 30/Box8B-S029-HCH Strata-X-C 500 mg/6 mL Tubes 30/Box8B-S029-EDG Strata-X-C 100 mg/12 mL Giga Tubes 20/Box8B-S029-HDG Strata-X-C 500 mg/12 mL Giga Tubes 20/Box8B-S029-JDG Strata-X-C 1 g/12 mL Giga Tubes 20/Box8E-S029-AGB Strata-X-C 96-Well Plate 10 mg/well 2/Box8E-S029-TGB Strata-X-C 96-Well Plate 30 mg/well 2/Box8B-S029-UGB Strata-X-C 96-Well Plate 60 mg/well 2/Box00F-4336-E0 Synergi Polar-RP 4 µm 150 x 4.6 mm ea00G-4375-Y0 Synergi Hydro-RP 4 µm 250 x 3.0 mm eaAH0-6023 12-Position SPE Vacuum Manifold ea

Gemini® 3 µm C18 (selected dimensions)*Part No. Dimension Unit00B-4439-B0 50 x 2.0 mm ea00B-4439-E0 50 x 4.6 mm ea00F-4439-B0 150 x 2.0 mm ea00F-4439-E0 150 x 4.6 mm ea

Gemini® 5 µm C18 (selected dimensions)*Part No. Description Unit00B-4435-B0 50 x 2.0 mm ea00F-4435-Y0 150 x 3.0 mm ea00F-4435-E0 150 x 4.6 mm ea00G-4435-E0 250 x 4.6 mm ea

* Other dimensions and particle sizes available, please contact your Phenomenex represen-tative for more information.

References1. 1. A. Posyniak, J. Zmudzki, J. Niedzielska, T. Sniegocki and A. Grzebalska,

APIACTA, 2003, 38, 249-256.

2. A. Kaufmann, S. Roth, B. Tyser, M. Widmer and D. Guggisberg, J. AOAC International, 2002, 85, 853-860.

3. S.Huq, J. Teuscher and K. Kallury, LC-GC (Europe) Applications Book, Sept. 2003, pp.16-17.

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I need to separate [compound/structure]

By: HPLC SPE GC Flash

Goal of separation

Sample Matrix

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Solid Phase ExtractionPart No. Description Unit Qty

8B-S029-TAK Strata-X-C 30 mg/1 mL Tubes 100/Box

8B-S029-UBJ Strata-X-C 60 mg/3 mL Tubes 50/Box

8B-S029-FBJ Strata-X-C 200 mg/3 mL Tubes 50/Box

8B-S029-HBJ Strata-X-C 500 mg/3 mL Tubes 50/Box

8B-S029-ECH Strata-X-C 100 mg/6 mL Tubes 30/Box

8B-S029-FCH Strata-X-C 200 mg/6 mL Tubes 30/Box

8B-S029-HCH Strata-X-C 500 mg/6 mL Tubes 30/Box

8B-S029-EDG Strata-X-C 100 mg/12 mL Giga Tubes 20/Box

8B-S029-HDG Strata-X-C 500 mg/12 mL Giga Tubes 20/Box

8B-S029-JDG Strata-X-C 1000 mg/12 mL Giga Tubes 20/Box

8E-S029-AGB Strata-X-C 96-Well Plate 10 mg/well 2/Box

8E-S029-TGB Strata-X-C 96-Well Plate 30 mg/well 2/Box

8B-S029-UGB Strata-X-C 96-Well Plate 60 mg/well 2/Box

Gemini® 3 µm C18Part No. Dimension Unit Qty

00B-4439-B0 50 x 2.0 mm ea

00B-4439-E0 50 x 4.6 mm ea

00F-4439-B0 150 x 2.0 mm ea

00F-4439-E0 150 x 4.6 mm ea

Gemini® 5 µm C18Part No. Description Unit Qty

00B-4435-B0 50 x 2.0 mm ea

00F-4435-Y0 150 x 3.0 mm ea

00F-4435-E0 150 x 4.6 mm ea

00G-4435-E0 250 x 4.6 mm ea

Luna® HILIC 3 µmPart No. Dimension Unit Qty

00B-4449-Y0 50 x 3.0 mm ea

00F-4449-Y0 150 x 3.0 mm ea

00D-4449-E0 100 x 4..6 mm ea

00F-4449-E0 150 x 4.6 mm ea

Luna® HILIC 5 µmPart No. Description Unit Qty

00F-4450-Y0 150 x 3.0 mm ea

00D-4450-E0 100 x 4..6 mm ea

00F-4450-E0 150 x 4.6 mm ea

00G-4450-E0 250 x 4.6 mm ea

Synergi™ Polar-RP 4 µm Part No. Description Unit Qty

00F-4336-E0 Synergi™ 4μm Polar-RP 150 x 4.6 mm ea

Synergi™ Hydro-RP 4 µm Part No. Description Unit Qty

00G-4375-Y0 Synergi Hydro-RP 4 µm 250 x 3.0 mm ea

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All trademarks have been acknowledged and are sole property of Phenomenex, Inc. © 2008 Phenomenex, Inc. All rights reserved.

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