UHPLC-MS/MS method for determination of atorvastatin calcium in human plasma: Application to a pharmacokinetic study based on healthy volunteers with specific genotype

Highlights

• A simple and sensitive determination method of atorvastatin calcium was developed and validated using UHPLC-MS/MS. The LLOQ was 0.05 ng/ml (S/N＞10).

• This method was successfully applied to a pharmacokinetic study in healthy volunteers.

• The genetic polymorphisms of SLCO1B1 521T＞C(rs4149056), SLCO1B1 388A＞G (rs2306283), CYP3A4 1*B(rs2740574), CYP3A4 1*G(rs2242480) and CYP3A5*3(rs776746) were analyzed by fluorescence in situ hybridization (FISH) technology in 187 healthy volunteers, respectively.

• This pharmacokinetic study was firstly based on healthy volunteers whose genotypes of SLCO1B1 521T＞C(rs4149056), SLCO1B1 388A＞G(rs2306283), CYP3A4 1*B(rs2740574), CYP3A4 1*G(rs2242480) and CYP3A5*3(rs776746) were all wild types.

• The C_max of AC in human volunteers with the specific genotype was nearly 10 times higher than that previous reported, indicating that genetic polymorphisms of these specific genotypes have significant influence on pharmacokinetics of atorvastatin.

Abstract

A rapid, selective and sensitive ultra high performance liquid chromatography coupled with tandem triple quaternary mass spectrometry (UHPLC-MS/MS) method was developed and validated for the quantitative determination of atorvastatin calcium (AC) in human plasma. Separation of AC and rosuvastatin calcium (internal standard, IS) were achieved on a Dikma Leapsil C18 reversed phase column (100 × 2.1 mm, 2.7 μm) with gradient elution using 0.2% (v/v) formic acid in water and acetonitrile as mobile phases, at the flow rate of 0.3 mL/min. AC and IS were detected using MS/MS with turbo ion pray source in negative mode by monitoring the precursor-to-product ion transitions m/z 557.0→453.0 for AC and m/z 480.0→418.0 for IS. The calibration curves were linear from 0.05 to 50 ng/mL with a correlation coefficient ( r²) of 0.9992 or better. Thereafter, 187 healthy candidates were checked to the genetic polymorphism analysis of SLCO1B1 521T＞C(rs4149056), SLCO1B1 388A＞G(rs2306283), CYP3A4 1*B(rs2740574), CYP3A4 1*G(rs2242480) and CYP3A5*3(rs776746) using fluorescence in situ hybridization technology. The genotype frequencies of wild-type homozygote, mutant heterozygote and mutant homozygote were 62.57%(TT), 34.22%(TC) and 3.21%(CC) for SLCO1B1 521T＞C, and 8.56%(AA), 33.69%(AG) and 57.75%(GG) for SLCO1B1 388A＞G, and 62.57%(CC), 34.22%(CT) and 3.21%(TT) for CYP3A4 1 G, and 58.29%(GG), 34.76%(GA) and 6.95%(AA) for CYP3A5*3, respectively. Furthermore, each tested genotype of CYP3A4 1B was wild type. Finally, 5 candidates with specific genotype described above were recruited to carry out the clinical pharmacokinetics of AC (n = 5). The validated UHPLC-MS/MS method was implemented in a high-throughput setting, capable of analyzing up to 288 samples per day, and was successfully applied to the pharmacokinetic study of AC based on healthy volunteers with specific genotype. The C_max of AC in human volunteers with the specific genotype was nearly 10 times higher than that previous reported, indicating that genetic polymorphisms of these specific genotypes have significant influence on pharmacokinetics of atorvastatin.

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.