UHPLC-MS/MS method for determination of atorvastatin calcium in human plasma: Application to a pharmacokinetic study based on healthy volunteers with specific genotype
Introduction
Atorvastatin, a synthetic and lipophilic statin, inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis, was recognized by the role in reducing the risk of heart and cerebrovascular events were widely used in clinical application [[1], [2], [3], [4], [5], [6]]. However, individual differences which cannot be ignored were existed in the clinical efficacy and the incidence of adverse events of atorvastatin. The critical factors causing the difference were the diverse genetic characteristics of drug metabolism and transport in vivo, especially the genetic polymorphism of the main metabolic enzymes and transporters of statins [[7], [8], [9], [10]]. In order to optimize the dosage regimen and minimize the risk of side effects, it is necessary to study the effect of genetic factors on clinical pharmacokinetics of atorvastatin.
Many analytical methods, such as UV spectrophotometric [11], HPLC-UV or HPLC-DAD [12,13], HPTLC [14], HPLC with fluorescence detection [15], have been reported for the determination of atorvastatin. However, these methods showed poor selectivity and time-consuming. What’s more, plenty of LC–MS/MS [[16], [17], [18], [19], [20], [21], [22], [23], [24], [25]] methods have been developed for the quantitation of atorvastatin individually or in combination with other drugs in biological matrices. The major limitations involving relatively high limit of quantitation(LOQ), longer chromatographic runtime, calling for a complex mobile phase, requiring a complex liquid-liquid extraction, and a narrow linearity range were shown in these methods [[16], [17], [18], [19], [20], [21], [22], [23], [24], [25]]. Interestingly, most of these LC–MS/MS methods were developed for the quantitation of atorvastatin with the molecular weight at 558 that is actually named atorvastatin acid (AA) [[16], [17], [18], [19], [20], [21], [22]]. Yacoub M et al. [16] determined atorvastatin in human plasma by protein precipitation with acetonitrile but with a high LOQ at 1.5 ng/ml. The chromatographic separation was achieved by the system which was adjusted by trichloroacetic acid. Wang J et al. [17] developed a method for simultaneous quantification of atorvastatin and other drugs but a long run time of 12 min was needed that is time-consuming in detecting huge number of analyzed samples for clinical application. The LOQ in this method was 0.1 nM that is equivalent to 0.12 ng/mL. Hotha K K et al. [19] determined atorvastatin and other drugs using liquid-liquid extraction but with a relatively higher LOQ at 0.20 ng/ml and a narrow linearity range within 0.2 ng/mL-30 mg/mL. Macwan J S et al. [20] reported a method, comparable in terms of sensitivity with the present work, with LOQ of 0.050 ng/mL for AA and its metabolites. Only several methods were developed for the quantitation of atorvastatin calcium (AC) with the molecular weight at 1209.42, which was actually the calcium salt of atorvastatin [23,[17], [18], [19], [20], [21], [22], [23], [24], [25]]. Zhou et al. [24] employed liquid-liquid extraction for sample preparation using a mixture of methyl tert-butyl ether and ethyl acetate as the extractant, which was quite complicated and time-consuming. The chromatographic separation was achieved on a complex mobile phase consisted of acetonitrile and ammonium acetate buffer (20 mM) containing 0.3% formic acid. Recently, Rezk M R [25] reported a method, comparable in terms of sensitivity with the present work, with LLOQ of 0.050 ng/mL for AC using liquid-liquid extraction for two times, which was quite tedious and time-consuming. It was worth mentioning that all these methods [16,17,19,[17], [18], [19], [20], [21], [22], [23], [24], [25]] were detected using electrospray ionization in positive mode by monitoring the precursor-to-product ion transitions m/z 559→440. In this research, AC (not AA), was detected by using UHPLC-MS/MS method with turbo ion pray source in negative mode by monitoring the precursor-to-product ion transitions m/z 557→397.
The genetic polymorphism of drug metabolism enzymes and transporters could significantly influence the pharmacokinetic process [26]. Genetic variations on drug transporter genes, SLCO1B1; P450 system genes, CYP3A4, CYP3A5, and CYP2D6 had been suggested to have associations with statin responsiveness [27]. For instance, Pasanen M K [28] found that SLCO1B1 polymorphism has a larger effect on the AUC of atorvastatin. Birmingham B K [29] verified that polymorphisms in SLCO1B1 T521 > C was associated with higher exposure to atorvastatin within a population. Becker M L [30] reported that the CYP3A4*1B G allele is associated with a lower risk of elevated statin plasma levels. Gao Y [31] reported that CYP3A4*1 G polymorphism is associated with lipid-lowering efficacy of atorvastatin.
To further study the genetic polymorphism of drug metabolism enzymes CYP3A4 and CYP3A5, and the transporter SLCO1B1 on the pharmacokinetics of AC, the current study used the fluorescent in situ hybridization (FISH) technique as an ancillary tool to investigate the genetic polymorphism of SLCO1B1 521T>C(rs4149056), SLCO1B1 388A>G(rs2306283), CYP3A4 1*B(rs2740574), CYP3A4 1*G(rs2242480) and CYP3A5*3(rs776746) in 187 healthy volunteers, respectively. And then pharmacokinetic study of AC was carried out based on healthy male volunteers whose genotypes of them were all wild type (n = 6). Suitable sample pretreatment method was developed and applied to multitudinous plasma samples.
Section snippets
Reagents and chemicals
Atorvastatin calcium (Lipitor, Pfizer China) was purchased from Beijing Luhe Hospital Affiliated to Capital Medical University. Chemical reference substances of atorvastatin calcium (purity, ≥98%),) and rosuvastatin calcium (purity, ≥98%) (Fig. 1) were obtained from national institute for food and drug control (Beijing, China). Acetonitrile and methanol were purchased from Fisher Scientific (Fair lawn, NJ, USA) as the HPLC grade. Formic acid was purchased from Dikma Reagent Company (Beijing,
Method development of UHPLC-MS/MS
Protein precipitation technique was tested for sample preparation in this study. The protein was commendable precipitated at a ratio of 1: 3 of plasma sample: precipitator (v: v). The precipitant composed of 50% acetonitrile and methanol (v:v) could provide a satisfactory recovery(>85% or better) of AC in human plasma. All the samples were respectively vortexed for 2 min and centrifugated at 14,000 rap/min at 4 °C for 15 min. Then each supernatant was filtered with a 0.22 μm of membrane and
Conclusions
A UHPLC-MS/MS method has been developed and validated for the quantitative determination of AC in human plasma, and has been successfully used for the pharmacokinetic study based on healthy volunteers with specific genotype. The performance criteria for the sensitivity, precision and accuracy, recovery and matrix effect, linearity and stability have been assessed and proved to meet the FDA’s requirements. Quantification was based on the IS method of plotting the peak areas ratios of the AC/IS
Author contributions
Binbin Xia made substantial contributions to the experiment, acquisition of data, analysis and interpretation of data, drafted and revised the article. Yali Li, Xiaorong Li, Yatong Zhang, Ming Xue, Pingxiang Xu, Tao Xia also participated in this study and gave suggestions for writing. Shicai Chen and Binbin Xia made main contributions to the conception and design, revised and discussed the experiments, revised the article and gave the final approval of the version to be submitted.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank the Beijing Municipal Natural Science Foundation (7163220), the Project of Basic Research and Clinical Application Collaboration Scientific Research of Capital Medical University (16JL-L06), the Tongzhou District Science and Technology Planning Project, Beijing (KJ2016CX037-23), the Tongzhou District Health Development Scientific Research Project, Beijing (TWKY-2016-QN-01-60), and the Medical Development Scientific Research Foundation Project of Beijing Luhe Hospital
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