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Highly Sensitive Determination of Colchicine in Human Plasma by UPLC-MS/MS for a Clinical Study in Healthy Subjects

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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, r 2 > 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.
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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: pranav_shrivastav@yahoo.com
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
67
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).
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
68
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.
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
69
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
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
70
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 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.
Jaivik V. Shah, Priyanka A. Shah, Daxesh P. Patel, Primal Sharma, Mallika Sanyal, and Pranav S. Shrivastav /
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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, AUC0120h
and AUC0inf 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
Jaivik V. Shah, Priyanka A. Shah, Daxesh P. Patel, Primal Sharma, Mallika Sanyal, and Pranav S. Shrivastav /
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73
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)
Accuracy (%)
System performance at LLOQ (0.01 ng/mL)
S/N ratio
Autosampler carry-over
Blank plasma area response
Method ruggedness
Precision (% CV)
Accuracy (%)
Dilution integrity
Precision (% CV)
Accuracy (%)
LLOQ and LOD (S/N ratio)
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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
Precision (% CV)
0.75-2.42
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
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
77
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
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
78
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;
100
samples 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
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
79
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
----
----
----
----
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
80
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)
Retentio
n 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)
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
81
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.
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
82
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Background: The labeling for colchicine (indicated for acute gout flares or prophylaxis) includes strict advisories regarding drug-drug and drug-food interactions, including warnings against consuming grapefruit or grapefruit juice during treatment. Two of the furocoumarins in grapefruit juice and Seville orange juice can inhibit intestinal cytochrome P450 (CYP) isozyme 3A4 and P-glycoprotein (involved in colchicine metabolism and transport). Severe toxicities in patients consuming these juices while taking other drugs metabolized through these pathways have been reported. Objective: Two Phase I studies assessed the effects of multiple daily consumptions of Seville orange juice or grapefruit juice on the pharmacokinetic properties of colchicine in healthy volunteers. Methods: Healthy volunteers were enrolled in 2 open-label, Phase I studies. Undiluted juice (240 mL) was administered twice daily for 4 days. Pharmacokinetic data were obtained following a single 0.6-mg dose of colchicine before the administration of juice and again following a single 0.6-mg dose of colchicine on the final day of juice administration. In each study, blood samples for pharmacokinetics were collected before dosing with colchicine and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 hours postdose. All subjects were monitored for adverse events (AEs) throughout the confinement portion of the study and were queried at the outpatient visits. AEs were coded according to corresponding MedDRA-coded system organ classes. Results: Forty-four subjects received either grapefruit juice (72.7% male; 90.9% white) or Seville orange juice (62.5% female; 100% white). Although it is considered to be a moderate concentration-dependent CYP3A4 inhibitor, grapefruit juice did not significantly affect the pharmacokinetic parameters of colchicine. When colchicine was administered with Seville orange juice, a moderate inhibitor, C(max) and AUC were decreased by ∼24% and ∼20%, respectively. Seville orange juice also caused, on average, a 1-hour delay in T(max). Colchicine in combination with grapefruit or Seville orange juice was well tolerated. There were no significant treatment-related AEs reported, and the most likely AEs were general gastrointestinal events. Conclusions: In contrast to label warnings based on the literature, grapefruit juice did not affect the pharmacokinetics of colchicine. Seville orange juice paradoxically reduced absorption of colchicine and increased T(max), but the clinical significance of this is unknown. Contrary to the expected effects of inhibiting the enzymes that metabolize colchicine, neither juice increased exposure to colchicine. However, the absence of a positive control in these studies dictates that caution should be used when applying these results clinically. ClinicalTrials.gov identifiers: NCT00960193 and NCT00984009.
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Background: Colchicine is a common drug used in inflammatory diseases. The narrow therapeutic index requires fast and reliable techniques for its quantitation. An online, automated sample preparation using TurboFlow™ technology combined with triple-stage quadrupole MS detection was applied to identify colchicine in human plasma and follow intoxications. Methodology: Plasma samples (200 µl) were mixed with deuterated colchicine and protein precipitation ZnSO4 solutions. After centrifugation, supernatants were extracted onto a Cyclone P TurboFlow column and eluted onto a narrowbore Hypersil™ GOLD column with a methanol/water gradient. Analytes were monitored in SRM mode (positive electrospray). Results: Total run time was 9.5 min. Calibration curves ranged from 0.342 to 17.1 ng/ml, with significant linearity (R(2) >0.99). Inter- and intra-assay precisions were <16.8% and accuracy was 84.4-110%. Conclusion: This method is suitable for monitoring intoxication in patients undergoing chronic treatment and is routinely applied to toxicological samples.
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Oral colchicine (Colcrys (R)) is approved in the US for the treatment of acute gout flares in adult patients and the prophylaxis of gout flares in patients aged >16 years. Colchicine is a tricyclic alkaloid that interrupts multiple inflammatory response pathways. Its principal mechanism of action in gout is thought to be inhibition of cytoskeletal microtubule polymerization, an important process in neutrophil functioning. In a phase III, randomized, double-blind, placebo-controlled, multicentre trial, the recommended dosage of Colcrys (R) (1.2 mg at the first sign of the flare, followed by 0.6 mg in 1 hour) was significantly more effective than placebo in treating acute gout flare, as assessed by the proportion of patients experiencing a >= 50% reduction in pain within 24 hours of initiating treatment. In a randomized, double-blind, placebo-controlled, single-centre trial, non-approved colchicine 0.6 mg once or twice daily (up to 6 months) was more effective than placebo in preventing gout flares in patients receiving allopurinol as urate-lowering therapy. At the recommended dosage for the treatment of acute gout flares, Colcrys (R) was as well tolerated as placebo in patients with gout. The incidence of the most common adverse events was similar between recipients of the recommended dosage of Colcrys (R) and placebo.
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Oral colchicine (Colcrys) is approved in the US for the treatment of acute gout flares in adult patients and the prophylaxis of gout flares in patients aged >16 years. Colchicine is a tricyclic alkaloid that interrupts multiple inflammatory response pathways. Its principal mechanism of action in gout is thought to be inhibition of cytoskeletal microtubule polymerization, an important process in neutrophil functioning. In a phase III, randomized, double-blind, placebo-controlled, multicentre trial, the recommended dosage of Colcrys (1.2 mg at the first sign of the flare, followed by 0.6 mg in 1 hour) was significantly more effective than placebo in treating acute gout flare, as assessed by the proportion of patients experiencing a >or=50% reduction in pain within 24 hours of initiating treatment. In a randomized, double-blind, placebo-controlled, single-centre trial, non-approved colchicine 0.6 mg once or twice daily (up to 6 months) was more effective than placebo in preventing gout flares in patients receiving allopurinol as urate-lowering therapy. At the recommended dosage for the treatment of acute gout flares, Colcrys was as well tolerated as placebo in patients with gout. The incidence of the most common adverse events was similar between recipients of the recommended dosage of Colcrys and placebo.