Development of a new HPLC method for simultaneous determination of clopidogrel and its major metabolite using a chemometric approach.

This paper presents the development and validation of a new HPLC-UV method for simultaneous quantitative determination of clopidogrel and its hydrolysis product clopidogrel carboxylic acid (CCA) from bulk material and dosage formulations. Development of the chromatographic method is based on a design of experiments (DOE) approach. A Box-Behnken experimental design was used to build the mathematical models and to choose the significant parameters for the optimization by simultaneously taking resolution, capacity factor and peak symmetry as responses. Derringer's desirability function was used for the selection of the optimum experimental conditions in terms of mobile phase composition, column temperature and flow rate. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, robustness and system suitability. The method was further employed for the study of clopidogrel bisulfate hydrolysis kinetics under different pH conditions, with special emphasis on the acidic hydrolysis studies, since different clopidogrel salts are suffering pre-systemic metabolism trough hydrolysis under the acidic pH of the stomach. This effect is generally difficult to quantify, since CCA is also the main circulating metabolite, and no differentiation between the pre-systemic and systemic CCA can be made. In the acidic environment created by a 0.1N HCl solution CLO degradation was slow at room or body temperature (25 and 37°C respectively), less than 5% of the initial CLO amount being hydrolyzed after 48h. Under forced conditions (85ºC) however, 17.8% of CLO was transformed into CCA within 48 hours.

Introduction

Clopidogrel (CLO) is a thienopyridine antiplatelet agent widely used in the management of different cardiovascular diseases, including atherothrombosis, unstable angina, or myocardial infarction [1]. The compound itself is a pro-drug which is inactive in vitro. In vivo, it selectively and irreversibly inhibits the binding of adenosine diphosphate (ADP) to its platelet receptors and the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex [2] following biotransformation to a thiolic active metabolite by means of CYP3A4 and CYP2C19 cytochrome P450 isoforms.[3].

Although CLO can be considered a ‘blockbuster' drug – the CLO innovator product ranks second worldwide in the top of incomes generated by a drug [4], large interindividual variability in clopidogrel response has been reported [5], leading to 20-40% of patients presenting “resistance” to treatment, associated with a low protection rate against atherothrombotic events [6]. Despite its widespread use, associated with significant “resistance” to treatment (for 20-40% of the patients). An important limitation in the study of CLO variability is the fact that until recently, no analytical methods were available for quantitative analysis of CLO and of its active thiol metabolite [7, 8, 9, 10]. The study of CLO pharmacokinetics was based for many years on the determination of its major but inactive circulating metabolite, clopidogrel carboxylic acid (CCA), resulted in vivo via hydrolysis of CLO mediated by the hepatic esterases (1) [11].

Figure 1

Chemical structure of CLO and of its hydrolytic degradation product and major in vivo metabolite, CCA

However, CCA is at the same time the main CLO impurity, since CLO hydrolysis can occur in different conditions, such as the acidic gastric environment. In the EMA's “Questions & Answers: Positions on specific questions addressed to the pharmacokinetics working party” [12], the EMA experts underline the fact that under fasting conditions, the dissolution in the gastric media with a subsequent hydrolysis and formation of the inactive CCA metabolite is maximal, and the amount of CLO available for absorption is reduced. Since CCA formed in the stomach is readily absorbable, no in vivo differentiation between CCA resulted from pre-systemic metabolism and the one resulting from hepatic hydrolysis can be performed. Therefore, an in vitro evaluation of the CLO hydrolysis kinetics arises as a necessity.

On the other hand, no HPLC method for simultaneous determination of CLO and CCA from bulk substance of pharmaceutical formulation is presented in literature. A literature survey revealed various assays for determining clopidogrel in pharmaceutical dosage forms, including spectrophotometry [13] thin-layer chromatography [14], high-performance thin-layer chromatography [15], HPLC-UV [16, 17, 18, 19, 20, 21, 22], LC-MS [23] or voltammetry [24]. CCA is determined usually from plasma samples, following its use as CLO “surrogate” in bioequivalence studies. Generally HPLC-UV [25], LC-MS [26, 27, 28] are used for separation and quantitative evaluation.

The present study aims to present the development and validation of a new HPLC-UV method for simultaneous quantitative evaluation of CLO and CCA from bulk material and pharmaceutical formulations, based on a design of experiments (DOE) approach.

The method is indented to quantitatively evaluate CLO degradation kinetics under different pH conditions, with special emphasis on CLO hydrolysis kinetics in simulated gastric fluid, in order to quantitatively evaluate the pre-systemic metabolism of CLO.

Material and Methods

Chemicals

Clopidogrel bisulfate standard was obtained from LGC Promochem GmbH (Germany). Clopidogrel Carboxylic Acid HCl was purchased from SynFine Research (Richmond Hill, Ontario, Canada).

HPLC grade acetonitrile was purchased from Merck KGaA (Darmstadt, Germany). Trifluoroacetic acid for HPLC was purchased from Sigma-Aldrich (St Louis, MO, SUA). Water for chromatography (resistivity minimum 18.2 MΩ and TOC maximum 30 ppb) was produced within the laboratory by means of a TKA GenPure system and used during the experiments. All other reagents were of analytical grade, purchased from different commercial suppliers and used without further purification.

Stock and working standard solutions

The stock solutions of CLO and CCA were prepared at a concentration of 500 µg/mL in acetonitrile. Working dilutions from stock solutions of the analytes were freshly prepared when needed. Spiked calibration standards in plasma have been prepared with both analytes together at the concentrations of: 0.125, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 µg/mL.

The quality control samples used in the accuracy and precision evaluation (QCs) were spiked with the analytes at the following levels: 4, 20 and 80 µg/mL.

Quantitative HPLC analysis

The quantitative analysis of the selected compounds was carried out using a Waters liquid chromatographic system (Waters, Milford, MA, USA) consisting of consisting of a 600 E Multisolvent Delivery System, Waters AF in line degasser , 486 UV tunable absorbance detector and Waters 717 plus autosampler. The output signal was monitored and processed using Empower software (Waters, Milford, MA, USA). The chromatographic separation was achieved on a Hypersil Gold, 5-μm 150 x 4 mm column (Thermo Fisher Scientific,Waltham, MA, USA). The injection volume was 10 μl and the UV detection wad performed at 210 nm. The elution was isocratic with mobile phase consisting in mixtures of acetonitrile and triflouroacetic acid. The exact composition of mobile phase, flow rate and column temperature were determined by means of experimental design.

Optimization of the HPLC method

Selection of the chromatographic column, as well as the aqueous component of the mobile phase was performed by using the one-factor-at-a-time (OFAT) approach. 0,1%, trifloroacetic acid, 0.1% phosphoric acid, as well as 0.025M phosphate buffer solution with pH ranging between 3.0 and 7.0 were tested. The optimum results both in terms of resolution and peak symmetry were obtained with trifluoroacetic acid.

A Box-Behnken design approach of response surface methodology (RSM) was further employed in order to study the individual and combined effects of three independent factors, i.e. the column temperature (X1), flow rate (X2) and percent of acetonitrile in the mobile phase (X3) on chromatographic resolution (Y1), asymmetry (Y2), capacity factor - k’ (Y3), and number of theoretical plates - N (Y4) of the CLO and CCA peaks.

The experimental design for the optimization along with statistical analysis of data was performed with Design-Expert 9.0 software, Trial Version (Stat Ease Stat-Ease, Inc., Minneapolis, MN, USA).

The design matrix for the Box-Behnken study was generated using three factors at three levels, including five replicates of the central point, resulting in a total of 17 analytical experiments. All experiments were performed in randomized order to minimize the effects of uncontrolled variables that may introduce a bias in the measurements.

The range and the levels of experimental variables investigated in this study are presented in 1.

Table 1

Factors and levels of Box-Behnnken experimental design

Factors	Code	Range and levels
		-1	0	+1
Column temperature (ºC)	X1	30	40	50
Flow (mL/min)	X2	0.8	1	1.2
% organic modifier (acetonitrile)	X3	24	28	32

Experimental data were fitted according to the following second-order polynomial equation calculated by multiple regression analysis:

where Y represents the measured response, b are coefficients calculated by multiple regression analysis, Xi represent the main effects of the independent variables, XiXj the interaction terms between variables, Xi2 quadratic expressions of the independent variables (included into the model in order investigate nonlinearity) and ε represents the random error.

The statistical analysis of the model was performed in the form of analysis of variance (ANOVA). The quality of the fitted model was expressed by the coefficient of determination (R2) and its statistical significance was checked by F-test and P-value test.

Validation of the HPLC method

The HPLC method was subjected to validation in accordance with the International Conference on Harmonization (ICH) regulations Q2(R1) [29] in terms of specificity, linearity, precision (repeatability and intermediate precision) and accuracy.

Assay specificity was evaluated in relation to interferences from the matrix components in the drug-free media.

The linearity assessment was performed for both CLO and CCA by using nine concentration levels, in the range 0.125-100 µg/mL. All analyses were performed in triplicate. The calibration curves and corresponding determination coefficients (R2) were calculated by least squares linear regression analysis. Detection limit (LOD) and quantitation limit (LOQ) were evaluated based on the signal-to-noise ratio. The concentrations yielding to signal-to-noise ratios of 3:1 and 10:1 were considered as LOD and LOQ, respectively.

Precision was evaluated for repeatability and intermediate reproducibility on spiked samples, at three different concentration levels (QClow, QCmedium and QChigh). Precision was assessed by means of RSD% values computed for absolute peak areas resulting from interpolation on the corresponding calibration curves. Repeatability study was achieved by injection of 5 replicates from a single prepared spiked plasma sample within a single day experimental session, whereas intermediate reproducibility was tested by means of five different samples processed in different experimental sessions for each concentration level.

The bias (%) between the concentration values determined for the QC samples and their nominal values was used as accuracy indicator.

Results and Discussion

According to BBD designs, a total of seventeen tests (including five replicates of the center point) were carried out in random order (2).

Table 2

Box-Behnken experimental design matrix of the selected independent variables and studied responses

Run no.	X1	X2	X3	Resolution</p> <p>(Y1)	Tailing factor (Y2)		k' (Y3)		Plate Count (Y4)
					CCA	CLO	CCA	CLO	CCA	CLO
1	40	1	28	13.05	1.27	1.13	1.87	5.80	3328	4878
2	40	1.2	32	9.52	1.27	1.17	1.00	2.99	2551	4096
3	50	0.8	28	14.17	1.25	1.09	2.58	7.59	3928	5479
4	40	0.8	24	17.03	1.23	1.07	3.88	12.79	4286	5742
5	40	1	28	13.08	1.27	1.11	1.87	5.77	3326	4923
6	50	1.2	28	12.34	1.24	1.11	1.37	4.50	3030	4554
7	40	1.2	24	14.86	1.23	1.11	2.13	7.55	3270	4720
8	30	0.8	28	14.04	1.29	1.15	2.65	7.75	3804	5404
9	40	1	28	13.03	1.27	1.13	1.86	5.76	3291	4861
10	40	1	28	13.01	1.27	1.14	1.89	5.84	3315	4838
11	50	1	32	10.42	1.28	1.14	1.37	3.78	2968	4584
12	30	1	32	10.27	1.30	1.20	1.40	3.85	2915	4433
13	50	1	24	15.80	1.21	1.06	2.72	9.31	3771	5096
14	30	1.2	28	11.99	1.26	1.18	1.38	4.55	2878	4334
15	30	1	24	15.66	1.27	1.14	2.81	9.57	3612	5070
16	40	1	28	13.07	1.26	1.13	1.88	5.81	3205	4881
17	40	0.8	32	11.37	1.30	1.15	2.02	5.22	3455	5228

Resolution