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Columbia International Publishing American Journal of Modern Chromatography (2014) Vol. 1 No. 1 pp. 67-83 doi:10.7726/ajmc.2014.1006

Research Article

______________________________________________________________________________________________________________________________ *Corresponding e-mail: [email protected] 1 Department of Chemistry, School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380009,

Gujarat, India 2 Department of Chemistry, St. Xavier’s College, Navrangpura, Ahmedabad 380009, Gujarat, India

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Highly Sensitive Determination of Colchicine in Human Plasma by UPLC-MS/MS for a

Clinical Study in Healthy Subjects

Jaivik V. Shah1, Priyanka A. Shah1, Daxesh P. Patel1, Primal Sharma1, Mallika Sanyal2, and Pranav S. Shrivastav1* Received 14 June 2014; Published online 3 January 2015 © The author(s) 2014. Published with open access at www.uscip.us

Abstract A rapid and sensitive method is described using solid-phase extraction and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) for the determination of colchicine in human plasma. Chromatography was performed on Waters Acquity UPLC BEH C8 (50 × 2.1 mm, 1.7 µm) column for the analysis of colchicine and colchicine-d6 using acetonitrile-4.0 mM ammonium formate in water (90:10, v/v) as the mobile phase. Detection and quantitation was done by multiple reaction monitoring for colchicine (m/z 400.3 → 358.3) and IS (m/z 406.3 → 362.3) on a triple quadrupole mass spectrometer in the positive ionization mode. A linear range from 0.010-10.0 ng/mL with correlation coefficient, r2 > 0.9996 was established for colchicine using 100µL plasma. Highly precise and quantitative recovery ranging from 100.2 to 101.1 % was obtained across four quality control samples. Matrix effect was assessed by post-column infusion, standard line slope and post extraction spike methods. Stability of colchicine in plasma was determined for different storage conditions like bench top, processed sample, freeze-thaw and long term. The method was applied to a bioequivalence study with 0.6 mg colchicine in 28 healthy volunteers. Assay reproducibility was ascertained by reanalysis of 129 subject samples. Keywords: Colchicine; Colchicine-d6; UPLC-ESI-MS/MS; Sensitive; Human plasma; Bioequivalence

1. Introduction Gout is a painful and progressive disease which can lead to joint destruction and deformity if not treated adequately. It is a medical condition characterized by recurrent attacks of acute inflammatory arthritis due to impaired metabolism of purines. This leads to hyperuricaemia, leading to accumulation of the metabolic end product urate in joints (Richette and Bardin, 2010).

http://en.wikipedia.org/wiki/Inflammatory_arthritis

Jaivik V. Shah, Priyanka A. Shah, Daxesh P. Patel, Primal Sharma, Mallika Sanyal, and Pranav S. Shrivastav / American Journal of Modern Chromatography (2014) Vol. 1 No. 1 pp. 67-83

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Oral colchicine (COL) is recommended for the treatment of acute gout flares in adults and also for prophylaxis of gout flares in young patients. COL is a tricyclic alkaloid found in flowering plants Colchicum autumnale (meadow saffron) and Gloriosa superb (glory lily) and has anti-inflammatory properties (Nuki, 2008). It interrupts multiple inflammatory response pathways and works by inhibiting cytoskeletal microtubule polymerization, an essential process in neutrophil functioning (Watson et al., 2012; Yang, 2010). COL is generally well tolerated when used at low doses and has a narrow therapeutic index. However, as it is a CYP3A4 and a P-glycoprotein substrate, any interaction with concomitantly administered CYP3A4 inhibitors or P-glycoprotein inhibitors can lead to significant increase in plasma concentration of COL. This condition results in severe toxicity with adverse events. The clinically recommended dose of COL is 1-2 mg. It has low serum protein binding (39 %), primarily to albumin with absolute bioavailability of about 45 %. The mean plasma concentration of 2.5 ng/mL is reached in ~1.5 h after oral administration of 0.6 mg COL. It is metabolized by cytochrome P450 (CYP) 3A4 in vitro into two primary metabolites, 2-O-demethyl colchicine and 3-O-demethyl colchicine. The total plasma concentration of these metabolites is less than 5 % of that of the parent drug (Mutual Pharmaceutical Company, COLCRYS® , Prescribing Information, 2012). As very low levels of COL are found in plasma and its likely toxicity issues, it is essential to develop highly sensitive and selective bioanalytical methods for COL to minimize the risk of drug buildup, for optimization of therapy and to reduce the frequency of adverse events. Several methods are reported for determination of COL in different biological matrices such as human plasma (Abe et al., 2006; Dehon et al., 1999; Jiang et al., 2007; Lhermitte et al., 1985; Tracqui et al., 1996; Wason et al., 2012), human serum (Samanidou et al., 2006), human blood (Tracqui et al., 1996; ), human urine (Clevenger et al., 1991; Lhermitte et al., 1985; Tracqui et al., 1996), human tissue (Dehon et al., 1999) and rat blood, liver and kidney (Fernandez et al., 1993). Majority of these methods are intended for forensic and toxicological studies and only few of them deal with the pharmacokinetic analysis (Jiang et al., 2007; Tracqui et al., 1996; Wason et al., 2012). A rapid and sensitive LC-MS/MS method has been developed for COL with a limit of quantitation of 0.050 ng/mL in human plasma and applied it to a pharmacokinetic study in healthy subjects (Jiang et al., 2007). Wason et al. (2012) studied the effects of grapefruit and Seville orange juices on the pharmacokinetics of COL in healthy subjects. Recently, Bourgogne et al. (2013) proposed an online LC-MS/MS method using automated TurboFlow™ technology for sample preparation and quantification of colchicine in human plasma. Thus far, there are no reports on the use of UPLC-MS/MS for the determination of COL in human plasma. In the present work a highly sensitive, selective and high throughput UPLC-MS/MS method has been developed and validated as per USFDA guidelines. The method offers higher sensitivity, and small turnaround time for analysis using 100µL human plasma for solid phase extraction. Picogram quantities of COL were determined from human plasma with acceptable accuracy and precision. Precise and quantitative recovery with minimal matrix effect was obtained at all quality control levels. The method was successfully applied for a bioequivalence study in healthy subjects and demonstrates satisfactory reproducibility through incurred sample reanalysis.


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2. Experimental

2.1 Chemicals and Materials

Reference materials of colchicine (COL, 99.34 %) and colchicine-d6 (IS, 99.10 %) were obtained from Clearsynth Labs (P) Ltd. (Mumbai, India). HPLC grade acetonitrile was procured from Mallinckrodt Baker, S.A.de C.V. (Estado de Mexico, Mexico) and ammonium formate was obtained from S.D. Fine Chemicals Ltd. (Mumbai, India). LiChrosep® DVB-HL (30 mg, 1 cc) solid phase extraction (SPE) cartridges were obtained from Phenomenex India (Hyderabad, India). Water used in the entire analysis was prepared using Milli-Q water purification system from Millipore (Bangalore, India). Blank human plasma was obtained from Supratech Micropath (Ahmedabad, India) and was stored at –20 C until use. 2.2 Liquid Chromatographic and Mass Spectrometric Conditions

A Waters Acquity UPLC system (MA, USA) consisting of binary solvent manager, sample manager and column manager was used for setting the reverse-phase liquid chromatographic conditions. The analysis of COL and IS was performed on a Waters Acquity UPLC BEH C8 (50 × 2.1 mm, 1.7 µm) column and maintained at 35 °C in a column oven. The mobile phase consisted of acetonitrile-4.0 mM ammonium formate (90:10, v/v). The flow rate of the mobile phase was kept at 0.300 mL/min. Ionization and detection of COL and IS was carried out on a Waters Quattro Premier XE (USA) triple quadrupole mass spectrometer, equipped with electro spray ionization and operating in positive ionization mode. The source dependent and compound dependent parameters optimized for COL and IS are shown in Table 1. Quadrupole 1 and 3 were maintained at unit mass resolution and MassLynx software version 4.1 was used to control all parameters of UPLC and MS. Table 1 MS/MS source dependant and compound dependant parameters for COL and COL-d6

Parameters Set value Cone gas flow (L/h) 70 Desolvation gas flow (L/h) 700 Capillary voltage (kV) 1.6 Source temperature (°C) 110 Desolvation temperature (°C) 380 Extractor voltage (V) 5 Pressure of collision gas (psi) 6500 Cone voltage (V) (a) COL (b) COL-d6

18 16

Collision energy (eV) (a) Colchicine (b) Colchicine-d6

33 35

Dwell time (ms) 100 Mode of analysis Positive MRM ion transition (m/z) (a) Colchicine (b) Colchicine-d6

400.3/358.3 406.3/362.3

MRM: Multiple reaction monitoring; COL: Colchicine


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2.3 Standard stock, calibration standards and quality control samples

The standard stock solution of COL (100 µg/mL) was prepared by dissolving requisite amount in methanol, while the working solution (1.0 µg/mL) was prepared in methanol:water (50:50, v/v). Calibration standards (CSs) and quality control (QC) samples were prepared by spiking blank plasma with working solutions. Stock solution (100 µg/mL) of the internal standard was prepared by dissolving 1 mg of colchicine-d6 in 10.0 mL of methanol. Its working solution (10 ng/mL) was prepared by appropriate dilution of the stock solution in methanol:water (50:50, v/v). Standard stock and working solutions used for spiking were stored in refrigerator at 5°C, while calibration standards and QC samples in plasma were kept at -70 °C until use. 2.4 Sample preparation protocol

Prior to analysis, all frozen subject samples, CSs and QC samples were thawed and allowed to equilibrate at room temperature. To an aliquot of 100 µL of spiked plasma sample, 40 µL of internal standard was added and vortexed for 10s. Further, 100 µL of water was added and vortexed for another 10 s. Samples were then centrifuged at 13148 × g for 5 min at 10 °C and loaded on LiChrosep® DVB-HL (30 mg, 1cc) cartridges, after conditioning with 1 mL methanol followed by 1 mL of 4.0 mM ammonium formate in water. Washing of samples was done with 2 × 1 mL of 10 % (v/v) methanol in water and subsequently the cartridges were dried for 1 min by applying nitrogen (1.72 x 105 Pa) at 2.4 L/min flow rate. Elution of analyte and IS was done using 500 µL of mobile phase into pre-labeled vials, followed by evaporation of solvent at 50 C. The dried residue was reconstituted with 100 µL of mobile phase, briefly vortexed and 10 µL was used for injection in the chromatographic system. 2.5 Procedures for Method Validation

The bioanalytical method was validated as per the USFDA guidelines (FDA, 2001) and was similar to the one described in our previous report (Gupta et al., 2013; Patel et al., 2013; Sharma et al., 2014). 2.5.1 System Suitability, System Performance and Auto-sampler carryover

System suitability test is done to authenticate optimum instrument performance (e.g., sensitivity and chromatographic retention) and is performed by analyzing a reference standard solution prior to running the analytical batch. In this test, six consecutive injections of aqueous standard mixture of COL (10 ng/mL, upper limit of quantitation) and IS (10 ng/mL) were injected at the start of each batch during method validation. The precision (% CV) in the measurement of area response and retention time was assessed. Additionally, the accuracy in the measurement of solution concentration was also evaluated. System performance was checked by calculating the signal to noise ratio for quantifying lower limit of quantitation (LLOQ, 0.01 ng/mL) sample. In this experiment, one extracted blank (without COL and IS) and one processed LLOQ sample with IS was injected at the beginning of each analytical batch. Autosampler carryover was evaluated by sequentially injecting aqueous standard of COL, mobile phase, extracted blank plasma, upper limit of quantitation (ULOQ) sample, extracted blank plasma, LLOQ sample and extracted blank plasma at the start of each batch.


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2.5.2 Selectivity, Linearity and Intra- and Inter-batch Accuracy and Precision

Selectivity of the method towards endogenous plasma matrix components was verified in eight batches (6 normal lots of K3EDTA, 1 haemolysed, and 1 lipemic) of blank human plasma. In addition, interference due to some commonly used medications like paracetamol, chlorpheniramine maleate, diclofenac, caffeine, acetylsalicylic acid and ibuprofen by human volunteers was also checked. Their working solutions (100 ng/mL) were prepared in the mobile phase and 10 µL was injected to check for any possible interference at the retention time of COL and IS. The linearity of the method was determined by analysis of five linearity curves containing ten non-zero concentrations. The area ratio response for COL/IS obtained from multiple reaction monitoring was used for regression analysis. The calibration curves were analyzed individually by using least square weighted (1/x2) linear regression. Intra-batch accuracy and precision was determined by analyzing six replicates of QC samples along with calibration curve standards on the same day. The inter-batch accuracy and precision were assessed by analyzing five precision and accuracy batches on three consecutive days. Sample injection reproducibility was also checked by re-injecting one entire validation batch. 2.5.3 Ion-suppression, Recovery and Matrix Effect

Assessment of ion suppression/enhancement was ascertained through post column analyte infusion. For this experiment, a standard solution containing COL (10 ng/mL) and IS was infused post column via a ‘T’ connector into the mobile phase at 5.0 µL/min employing an infusion pump. Thereafter, aliquots of 10 µL of extracted control (blank) plasma were then injected into the column and multiple reaction monitoring (MRM) chromatograms were acquired for COL and IS. In MRM mode, two stages of mass filtering are employed on a triple quadrupole mass spectrometer. In the first stage, the precursor ion (m/z 400.3 for COL and m/z 406.3 for colchicine-d6) was preselected in Q1 MS and then induced to fragment by collisional excitation with nitrogen gas in a pressurized collision cell (Q2). In the second stage, instead of obtaining full scan ms/ms where all the possible fragment ions derived from the precursor ion in Q3 MS, only the most stable and consistent product ion (m/z 358.3 for COL and m/z 362.3 for colchine-d6) was analyzed. This targeted MS analysis using MRM enhances the detection limit for the analyte by several folds. The recovery of COL and IS after SPE was estimated by comparing the mean area ratio response of samples spiked before extraction to that of extracts with post-spiked samples (spiked after extraction) at four QC levels. Matrix effect, expressed as matrix factors (MFs) was assessed by comparing the mean area ratio response of post-extraction spiked samples with mean area ratio response of solutions prepared in mobile phase solutions (neat standards). For assays which are very selective it is unlikely that co-eluting peaks can impact the quantification of the analytes. Nevertheless, the presence of unmonitored, co-eluting compounds from the matrix can directly affect the accuracy, precision, ruggedness and overall reliability of a validated method. It is recommended that evaluation of MFs can help to assess the matrix effect (Bansal and DeStefano, 2007). MFs can be determined from the peak area response for the analyte and IS separately, while the ratio of the two factors yields IS-normalized MF for the analyte. The IS-normalized MFs using stable-isotope labeled IS should be close to unity due similarities in the chemical properties and elution times for the analyte and IS. IS-normalized MFs (COL/IS) were calculated to access the variability of the assay due to matrix effects. Relative matrix effect was assessed from the precision


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(% CV) values of the slopes of the calibration curves prepared from eight different plasma lots/sources. To prove the absence of matrix effect, % CV should be less than 3-4 % as recommended (Matuszewski, 2006) for method applicability to support clinical studies. Further, when using stable isotope-labeled drugs as internal standards the precision of standard line slopes should be ≤ 2.4 %. Additionally, the matrix effect on analyte quantification was also studied in the same ten batches/lots of plasma by preparing six replicates samples at LQC-1 and HQC levels (spiked after extraction) and checked for the % accuracy and precision (%CV). 2.5.4 Stability, Dilution Reliability and Method Ruggedness

The standard stock solutions of COL and IS were evaluated for short term and long term stability at 25 °C and 5 °C respectively. The analyte stability in spiked plasma samples was evaluated by measuring the area ratio response (COL/IS) of stability samples against freshly prepared standards having identical concentration. Bench top (at room temperature), processed sample stability at room temperature and at refrigerated temperature (5 °C), dry extract, freeze-thaw (-20 °C and -70 °C) and long term (-20 °C and -70 °C) stability of COL in plasma was studied at two QC levels. Method ruggedness study was intended to check for method reproducibility with the same samples (using two precision and accuracy batches) from analyst to analyst and column to column (two different columns of the same make having different batch numbers), while keeping the optimized method parameters constant. The degree of reproducibility was evaluated and expressed in terms of precision (% CV). Dilution integrity experiment was evaluated by diluting the stock solution prepared as spiked standard at 20 ng/mL COL concentration in the screened plasma. The precision and accuracy for dilution integrity standards at 1/5th (4 ng/mL) and 1/10th (2 ng/mL) dilution were determined by analyzing the samples against freshly prepared calibration curve standards. 2.6 Application of the method and incurred sample reanalysis (ISR)

A bioequivalence study was performed with a single dose of a test (0.6mg colchicine tablets from an Indian Pharmaceutical Company) and a reference (COLCRYS® , 0.6mg colchicine tablets from Mutual Pharmaceutical Company, Inc., Philadelphia, USA) formulation to 28 healthy Indian subjects under fasting. Each subject was judged to be in good health through medical history, physical examination and routine laboratory tests. All the subjects were informed about the objectives and possible risks involved in the study and a written consent was obtained. The entire study was as per the guidelines laid down by International Conference on Harmonization, E6 Good Clinical Practice (FDA, 1996). The subjects were orally administered a single dose of test and reference formulations after recommended wash out period of 7 days with 240 mL of water. Blood samples were collected at 0.00 (pre-dose), 0.16, 0.33, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.50, 4.00, 5.00, 6.00, 8.00, 10.0, 12.0, 16.0, 24.0, 36.0, 48.0, 72.0, 96.0 and 120 h after oral administration. During study, subjects had a standard diet while water intake was unmonitored. The pharmacokinetic parameters of COL were estimated by non-compartmental model using WinNonlin software version 5.2.1 (Pharsight Corporation, Sunnyvale, CA, USA). To determine whether the test and reference formulations were pharmacokinetically equivalent, Cmax, AUC0–120h and AUC0–inf and their ratios (test/ reference) using log transformed data were assessed. The drug formulations were considered pharmacokinetically equivalent if the difference between the compared parameters was statistically non-significant (p ≥ 0.05) and the 90% confidence intervals for these parameters were within 80-125%. An incurred sample reanalysis was also done by


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reanalysis of 129 subject samples. The selection criteria included samples which were near the Cmax and the elimination phase in the pharmacokinetic profile of the drug.

3. Results and Discussion

3.1 UPLC-MS/MS method development

Mass spectrometry: As COL is present in very low concentrations in blood, development of sensitivity and selective assay is a major criterion for its reliable estimation in plasma samples. Due to the presence of secondary nitrogen atom in COL, the present study was conducted using electrospray ionization (ESI) in the positive ionization mode for UPLC-MS/MS analyses to attain high sensitivity and a good linearity in the regression curves. The conditions for ESI of COL and IS were set so as to have predominant protonated precursor [M+H]+ ions which were found at m/z 400.3 and 406.3 respectively in the Q1 MS full scan spectra. The most abundant product ions in Q3 MS spectra for COL and IS were observed at m/z 358.3 and 362.3 respectively by applying 33 and 35 eV collision energy. These product ions were obtained by the elimination of an acetyl group from the precursor ions as shown in Fig. 1. A dwell time of 100 ms for both the compounds was adequate to obtain sufficient data points for quantification.

Fig. 1. Product ion mass spectra of (a) colchicine (m/z 400.3 → 358.3, scan range 50-450 amu) and (b) internal standard, colchicine-d6 (m/z 406.3 → 362.3, scan range 50-450 amu) in positive ionization mode

Plasma extraction: Several reported assays have used liquid-liquid extraction (LLE) for sample clean-up from different matrices (Abe et al., 2006; Fernandez et al., 1993; Jiang et al., 2007; Lhermitte et al., 1985; Tracqui et al., 1996). Mainly dichloromethane (DCM) has been employed for quantitative recovery of COL in these procedures. Thus, LLE was tried initially using DCM and methyl tert-butyl ether solvents, alone and also in combination (different volume ratios, 50:50, 60:40 and 70:30, v/v). In all the trials, the recovery ranged from 73-89 %, however, it was difficult


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to obtain consistent results at LLOQ and low quality control (LQC) samples. Thus, SPE was explored which has been reported by Wason et al. (2012) for sample preparation. LiChrosep® DVB-HL (30 mg, 1 cc) SPE cartridges were used to obtain quantitative and precise recovery at all QC levels. To improve the extraction recovery, washing and elution steps were suitably optimized for selective extraction of COL and IS from plasma matrix components. The mean extraction recovery of COL obtained (100.6 %) was highly consistent at all QC levels.

Fig. 2. MRM ion-chromatograms of (a) double blank plasma (without IS) (b) blank plasma with colchicine-d6 (IS), (c) colchicine at LLOQ and IS (d) subject sample at Cmax after administration of 0.6 mg dose of colchicine


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Chromatography: Chromatography was initiated on Waters Acquity UPLC BEH C8 (50 × 2.1 mm, 1.7 µm) column to achieve a short run time, good peak shapes, higher response, minimum matrix interference and solvent consumption. To find the best eluting solvent system, various combinations of methanol/acetonitrile with ammonium acetate/formate and formic acid in different volume ratios were tested. It was observed that there was a significant increase in the sensitivity (1.5 times) in presence of ammonium formate compared to ammonium acetate together with acetonitrile as the organic modifier. Acetonitrile has low ion suppression effect, is volatile, and therefore more compatible with MS detection. Best chromatographic conditions with respect to analyte response (greater signal to noise ratio) and peak shape were obtained with acetonitrile-4.0 mM ammonium formate (90:10, v/v) as the mobile phase at a flow rate of 0.300 mL/min. The MRM chromatograms of double black plasma (without analyte and IS), blank plasma spiked with IS, LLOQ sample and subject sample at Cmax concentration indicate absence of any interfering peaks at the retention of the analyte or IS (Fig. 2). The retention time for COL and IS were 1.04 and 1.05 min respectively in a run time of 1.5 min, which ensured high throughput of the method. The reproducibility of retention times for COL, expressed as % CV was ≤ 0.9 % for a minimum of 100 injections on the same column. The use of deuterated internal standard, colchicine-d6 helped in achieving overall accuracy and precision of the data. Use of internal standards in quantitative bioanalysis with mass detection helps to compensate any random and systematic errors due to changes in detector sensitivity (detector drift). MRM based methods, in principal, provide both absolute structural specificity for the analyte and relative or absolute measurement of analyte concentration when stable, isotopically-labeled standards are added to a sample in known quantities. When such internal standards are used, the concentration can be measured by comparing the signals from the exogenous labeled and endogenous unlabeled analyte as they possess same physicochemical properties and differ only by mass.

3.2 Validation Results

3.2.1 Assay performance and Method Selectivity

Table 2 UPLC-MS/MS assay performance for colchicines

System suitability with 10 ng/mL COL (n=6)

Precision (% CV) 0.15 % for retention time and 0.34 % for area response

Accuracy (%) Varied from 99.4-100.5 %

System performance at LLOQ (0.01 ng/mL)

S/N ratio ≥ 25 (% CV less than 20)

Autosampler carry-over

Blank plasma area response ≤ 2.235 (≤ 0.29 % of LLOQ response)

Method ruggedness

Precision (% CV) 0.62-1.09

Accuracy (%) Within 97.2-102.5 %

Dilution integrity


Accuracy (%) Within 98.4-101.6 %

LLOQ and LOD (S/N ratio) 10.0 pg/mL ( ≥ 25) and 3.5 pg/mL (≥ 11)


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The results for system suitability, system performance and auto-sampler carryover suggests acceptable assay performance as evident from the data presented in Table 2. The selectivity of the method is apparent from the chromatograms of blank plasma spiked with IS, analyte at LLOQ level and in subject sample at Cmax in Fig. 2. No interference due to endogenous components was found at the retention time of COL and IS. Additionally, none of the commonly used medications by human volunteers interfered at their respective retention times. 3.2.2 Linearity, Intra- and Inter-batch Accuracy and Precision The standard curves showed good linearity over the established concentration range of 0.010-10.0 ng/mL (r2 ≥ 0.9996) for COL. The mean regression lines, accuracy and precision data in the measurement of calibrator concentrations are shown in Table 3. The intra-batch precision (% CV) ranged from 2.12-4.14 % and the accuracy was within 99.0-102.0 % for COL. Similarly for inter-batch experiments, the precision varied from 1.24-5.84 % and the accuracy was within 98.5-101.0 % (Table 4). Table 3 Linearity assessment for colchicines

Linearity range (ng/mL) 0.010-10.0

Calibration standards (ng/mL) 0.010, 0.020, 0.050, 0.100, 0.200, 0.500, 1.00, 2.00, 5.00 & 10.0 Quality control samples (ng/mL) 0.010, 0.030, 0.360, 4.00 and 8.00

Weighting factor 1/x2

Mean regression line (y=mx + c) y = (0.2269 ± 0.0017) x + (0.000105 ± 0.000005)

Correlation coefficient (r2) 0.9996


Accuracy (% change) 98.6-101.5 %

Table 4 Intra- and inter-batch precision and accuracy for colchicines

Nominal concentration (ng/mL)

Intra-batch Inter-batch

Mean conc. found (ng/mL)a

% CV % Accuracy Mean conc. found (ng/mL)b

% CV % Accuracy

HQC (8.00) 8.16 2.12 102.0 8.01 1.24 100.1

MQC-1 (4.00) 3.97 2.28 99.3 3.94 1.99 98.5

MQC-2 (0.360) 0.364 2.26 101.1 0.367 2.83 100.2

LQC (0.030) 0.0301 3.82 100.3 0.0298 3.75 99.3

LLOQ QC (0.010) 0.0099 4.14 99.0 0.0101 5.84 101.0

CV: Coefficient of variation; n: Number of replicates; LQC: low quality control; MQC: medium quality control; HQC: high quality control; LLOQ QC: lower limit of quantitation quality control


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3.2.3 Recovery, Matrix Effect and Ion-suppression The extraction recovery and IS-normalized MF for COL are presented in Table 5. The mean extraction recovery varied from 100.2 to 101.1 % across QC levels. As presence of unmonitored, co-eluting compounds from the matrix can directly impact the overall reliability of a validated method, it is recommended to evaluate MFs to consider the matrix effect. Additionally, it is required to check the matrix effect in lipemic and haemolysed plasma samples together with normal K3EDTA plasma. The IS-normalized MFs using stable-isotope labeled IS should be close to unity due to similarities in the chemical properties and elution behavior of the analytes and ISs. The IS-normalized MFs ranged from 1.009-1.033 for COL. Table 5 Extraction recovery and matrix factors for colchicines

QC

level

Mean area ratio response (n = 6) Extraction

recovery

(C/B × 100)

Matrix factor

A B C Analyte

(B/A) IS

IS-

normalized

LQC 0.0068 0.0070 0.0071 101.1 (95.0)a 1.038 1.005 1.033

MQC-2 0.0856 0.0876 0.0882 100.7 (96.1)a 1.023 0.998 1.025

MQC-1 0.9336 0.9542 0.9559 100.2 (95.6)a 1.022 1.012 1.009

HQC 1.9721 2.0145 2.0219 100.4 (96.7)a 1.021 1.009 1.012

A: mean area ratio response of samples prepared by spiking in mobile phase (neat samples) B: mean area ratio response of samples prepared by spiking in extracted blank plasma C: mean area ratio response of samples prepared by spiking before extraction LQC: low quality control; MQC: medium quality control; HQC: high quality control; IS: internal standard; avalues for internal standard

Fig. 3. Injection of extracted blank human plasma during post column infusion of (a) colchicine and (b) colchicine-d6 at upper limit of quantitation (10 ng/mL) level In addition, interferences due to endogenous plasma components were also assessed by plotting calibration curves for eight different batches of blank plasma lots. The coefficient of variation (% CV) of the slopes of calibration lines for relative matrix effect in eight different plasma lots was 1.68. Further, the extracts obtained through the optimized SPE showed negligible matrix effect, which


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were analyzed by the post column analyte infusion method. The result confirmed the absence of signal suppression or enhancement at the retention time of COL and IS (Fig. 3). 3.2.4 Stability, Dilution Reliability and Method Ruggedness Stock solutions kept for short-term and long-term stability as well as spiked plasma solutions showed no evidence of degradation under all studied conditions. Samples for short-term stability remained stable upto 28 h, while the stock solutions of COL and IS were stable for minimum of 48 days at refrigerated temperature of 5 °C. No significant degradation was observed for both the analytes during sample storage and any of the processing steps during extraction. The detailed results for stability studies are presented in Table 6. The precision (% CV) values for dilution reliability were between 0.82 and 1.32 for both the dilutions. The precision and accuracy for method ruggedness on two different UPLC BEH C8 (50 × 2.1 mm, 1.7 µm) columns and with different analysts were within 0.62-1.09 % and 97.2-102.5 % respectively (Table 2).

Table 6 Stability of colchicine in plasma under various conditions (n = 6)

Storage conditions

Nominal concentration (ng/mL)

Mean stability sample (ng/mL) ± SD

% Change

Bench top stability at 25 °C, 20 h 8.00 8.04 ± 0.059 0.50 0.030 0.031 ± 0.001 2.00

Freeze & thaw stability at -20 °C 8.00 7.99 ± 0.084 -0.13 0.030 0.029 ± 0.001 -1.67

Freeze & thaw stability at -70 °C 8.00 7.97 ± 0.038 -1.13 0.030 0.029 ± 0.002 -1.33

Auto-sampler stability at 4°C, 36 h 8.00 8.02 ± 0.051 0.25 0.030 0.030 ± 0.003 0.33

Dry extract stability at 2-8°C, 24 h 8.00 8.02 ± 0.051 0.25 0.030 0.030 ± 0.003 0.33

Wet extract stability at 24 °C, 30 h 8.00 8.04 ± 0.063 0.52 0.030 0.029 ± 0.048 -1.07

Long term stability at -20 °C, 176 days 8.00 8.01 ± 0.081 0.13 0.030 0.031± 0.001 1.67

Long term stability at -70 °C, 176 days 8.00 8.06 ± 0.146 0.75 0.030 0.030± 0.001 1.33

SD: Standard deviation, n: Number of replicates;

100samples comparisonMean

samples comparisonMean ? samplesstability Mean %Change

3.3 Application to a bioequivalence study and ISR results

So far there are no reports on the pharmacokinetics of COL in Indian subjects. Therefore the method was applied to monitor COL concentration in human plasma samples after oral administration of a single 0.6 mg dose. Fig. 4 shows the mean plasma concentration vs. time profile for COL under fasting for test and reference formulations. Such a profile can be obtained by measuring the concentration of COL in plasma samples taken at specific time intervals after oral administration of the dose and plotting the concentration of drug in plasma against the


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corresponding time at which the plasma samples were collected. Five important pharmacokinetic parameters can be used to describe such a plasma level-time curve, which are useful in assessing the bioavailability of a drug from its formulation. This includes peak plasma concentration (Cmax), time of peak plasma concentration (Tmax), area under the curve (AUC), elimination half-life (t1/2) and rate constant (Kel). Table 7 summarizes the mean pharmacokinetic parameters obtained for COL after oral administration of test and reference formulations. The mean values for peak plasma concentration (Cmax, 4.21 ng/mL) for COL was achieved at ~1.5 h as reported in literature (Mutual Pharmaceutical Company, COLCRYS® , Prescribing Information, 2012). The ratios of mean log-transformed parameters and their 90% confidence intervals varied from 91.5 to 104.3 % for COL, which is within the acceptance range of 80-125 %. These results indicate that the rate and extent of drug absorption of COL is similar for the test and reference formulations. It has been shown that food is not associated with any clinically significant effects on the absorption of COL (Richette and Bardin, 2010).

Fig. 4. Mean plasma concentration-time profile of colchicine after oral administration of 0.6 mg (test and reference) tablet formulation to 28 healthy Indian volunteers

The blood sampling schedule was extended up to 120 h for accurate assessment of total AUC and to check for any secondary peak in COL plasma concentration, which could be due to reabsorption and/or biliary circulation of the drug as reported previously (Mutual Pharmaceutical Company, COLCRYS® , Prescribing Information, 2012).

Table 7 Mean pharmacokinetic parameters (Mean ±SD) and comparison of treatment ratios and 90% CIs of natural log (Ln)-transformed parameters

Parameter Test (T)

Reference (R)

Ratio (T/R) %

90% CI (lower – upper)

Power

Intra subject variation, (% CV)

Cmax (ng/mL) 4.21 ± 1.325 4.30 ± 1.562 97.6 91.5-103.8 0.9996 7.26 AUC0-120h(h. ng/mL) 18.32 ± 7.05 18.59 ± 6.43 98.3 94.4-104.3 0.9998 4.65 AUC 0-inf (h. ng/mL) 19.05 ± 6.24 19.36 ± 6.05 98.4 95.2-101.8 0.9993 2.86 Tmax (h) 1.48 ± 0.28 1.54 ± 0.32 ---- ---- ---- ---- t1/2 (h) 4.59 ± 1.02 4.35 ± 1.23 ---- ---- ---- ---- Kel (1/h) 0.151± 0.004 0.159 ± 0.004 ---- ---- ---- ----


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Cmax: Maximum plasma concentration; AUC0-t: Area under the plasma concentration-time curve from zero hour to 120 h; AUC0-inf: Area under the plasma concentration-time curve from zero hour to infinity; Tmax: Time point of maximum plasma concentration; t1/2: Half life of drug elimination during the terminal phase; Kel: Elimination rate constant; SD: Standard deviation; CI: confidence interval; CV: coefficient of variation The percentage change in analysing 129 incurred samples for assay reproducibility was within ± 14%, which is well within the acceptance criterion of ±20% (Yadav and Shrivastav, 2011). This further reinforces the reproducibility of the proposed method.

3.4 Comparison with existing methods

The proposed method is more sensitive and rapid (analysis time for extraction and chromatography) compared to all other procedures for determination of COL in different biological matrices. The present method employs small plasma volume (100 µL) for processing, which is much less compared to several reported methods. Furthermore, the chromatographic analysis time, on-column loading of COL at ULOQ and organic solvent consumption was significantly lower in comparison with reported assays. A detailed comparison of salient features of different chromatographic methods developed for COL is given in Table 8. Table 8 Comparative assessment of chromatographic methods developed for colchicine in

biological matrices

Detection technique

Extraction procedure

Sample volume

Linear range (ng/mL)

Retention time; run time

Application Reference

HPLC-UV

LLE 1000 µL human plasma & 20 mL urine

5.0-100 for plasma and 0.25-5.0 for urine

3.5 min; 8.0 min

Analysis of COL in plasma and urine samples of a poisoned subject

Lhermitte et al. (1985)

HPLC-UV LLE 200 µL rat plasma; 10 g liver and kidney

1000-5000 5.33 min; 9.0 min

Determination of COL levels in blood, liver and kidney of rats following intraperitoneal injection of 10 mg/kg of the drug

Fernandez et al. (1993)

LC-Ionspray-MS

LLE 4000 µL human blood, plasma and urine

5.0-200 2.70 min; 10 min

Analysis of blood sample from a subject who died due to COL overdose

Tracqui et al. (1996)

HPLC-UV

PP 100 µL human serum or urine

50-2500

5.0 min; 6.0 min

Determination of COL in commercial pharmaceuticals and biological fluids

Samanidou et al. (2006)


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LC-ESI-MS/MS

LLE 1000 µL human plasma

0.50-50 2.4 min; 6.0 min

Quantification of COL in postmortem samples

Abe et al. (2006)

LC-ESI- MS/MS

LLE 100 µL human plasma

0.05-10.0

1.99 min; 2.4 min

Pharmacokinetic study with 2.0 mg COL in 20 healthy subjects

Jiang et al. (2007)

LC-MS/MS

SPE -- 0.20-40.0 -- To study the effect of grapefruit and Seville orange juices on the pharmacokinetics of COL in 44 healthy subjects

Wason et al. (2012)

Turbulent flow LC-MS/MS

PP 200 µL human plasma

0.342-17.1 ~5.1 min; 9.5 min

Analysis of COL plasma samples obtained from a patient after voluntary intoxication

Bourgogne et al. (2013)

UPLC-MS/MS

SPE 100 µL human plasma

0.01-10.0 1.04 min; 1.50 min

Bioequivalence study with 0.6 mg oral dose of COL in 28 human subjects; ISR of 129 sample, % change within ± 14 %

Present work

LLE: liquid-liquid extraction; SPE: solid phase extraction; PP: protein precipitation; ISR: incurred sample reanalysis; COL: colchicine

4. Conclusion The proposed UPLC-MS/MS assay for the quantitation of colchicine in human plasma was developed and fully validated as per USFDA guidelines. The method offers significant advantages over those previously reported, in terms of lower sample requirements, simplicity of extraction procedure and overall analysis time. With dilution integrity up to two folds, it is possible to extend the upper limit of quantification up to 20 ng/mL. In addition, the autosampler carryover test, assessment of matrix effect (matrix factors and relative matrix effect in different plasma lots, post column analyte infusion), effect of commonly used medications by subjects and assay reproducibility is also studied in the present work. The developed method can be readily employed for in clinical and toxicological studies.

Acknowledgements The authors would like to thank the Department of Chemistry, Gujarat University for supporting this work.


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