Validation and application of a high-performance liquid chromatography–tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid–liquid extraction

doi:10.1016/j.chroma.2006.07.071

Journal of Chromatography A

Volume 1130, Issue 2, 20 October 2006, Pages 302-307

https://doi.org/10.1016/j.chroma.2006.07.071 Get rights and content

Abstract

Kaletra is an important antiretroviral drug, which has been developed by Abbott Laboratories. It is composed of lopinavir (low-pin-a-veer) and ritonavir (ri-toe-na-veer). Both have been proved to be human immunodeficiency virus (HIV) protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1 infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of lopinavir and ritonavir in human plasma, in which lopinavir and ritonavir can be simultaneously analyzed with high throughput. The sample preparation consisted of liquid–liquid extraction with a mixture of hexane: ethyl acetate (1:1, v/v), using 100 μL of plasma. Chromatographic separation was performed on a Waters Symmetry C₁₈ column (150 mm × 3.9 mm, particle size 5 μm) with reverse-phase isocratic using mobile phase of 70:30 (v/v) acetonitrile: 2 mM ammonium acetate aqueous solution containing 0.01% formic acid (v/v) at a flow rate of 1.0 mL/min. A Waters symmetry C₁₈ guard column (20 mm × 3.9 mm, particle size 5 μm) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of tetrahydrofuran (THF), methanol and water (45:45:10, v/v/v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over the concentration ranges of 19–5300 ng/mL for lopinavir and 11–3100 ng/mL for ritonavir. A-86093 was used as an internal standard (I.S.). The relative standard deviation (RSD) were <6% for both lopinavir and ritonavir. Mean accuracies were between the designed limits (±15%). The robust and rapid LC/MS/MS assay has been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.

Introduction

Kaletra is a co-formulation of lopinavir and ritonavir. Lopinavir is an inhibitor of HIV protease. As co-formulated in Kaletra, ritonavir inhibits the CYP3A-mediated metabolism of lopinavir, thereby providing increased plasma levels of lopinavir.

Lopinavir is chemically designed as [1S-[1R*), (R*), 3R*, 4R*]]-N-[4-[[(2,6-dimethylphenoxy)acetyl]amino]-3-hydrox-5-phenyl-1-(phenylmethyl)pentyl]tetrahydro-alpha-(1-methylethyl)-2-oxo-1(2H)-pyrimidineacetamide. Its molecular formula is C₃₇H₄₈N₄O₅, and its molecular weight is 628.80. Fig. 1 shows lopinavir structure.

Ritonavir is chemically designed as 10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-diozo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid, 5-thiazolylmethyl ester [5S-(5R*,10R*,11R*)]. Its molecular formula is C₃₇H₄₈N₆O₅S₂, and its molecular weight is 720.95. Ritonavir has the structural formula in Fig. 1.

Both lopinavir and ritonavir are white to light tan powder. They are freely soluble in methanol and ethanol, soluble in isopropanol and practically insoluble in water.

The combination of lopinavir and ritonavir is used in the treatment of the infection caused by the HIV, which is the virus responsible for acquired immunodeficiency syndrome (AIDS). The combination of lopinavir and ritonavir is used to slow the progression of disease in patients infected with HIV who have advanced symptoms, early symptoms, or no symptoms at all.

Lopinavir and ritonavir will not cure or prevent HIV infection or AIDS; however, it helps to keep HIV from reproducing and appears to slow down the destruction of the immune system. This may help delaying the development of problems usually related to AIDS or HIV disease. Lopinavir and ritonavir will not keep HIV affected from spreading HIV to other people. People who receive this medicine may continue to have other problems usually related to AIDS or HIV disease.

The ever-shortening timeline in drug discovery and development have brought about the need for high throughput approaches to methods used to quantitate drugs, metabolites and endogenous biomolecules in biological matrices [1], [2], [3]. A rapid and high-throughput method has been developed and validated to simultaneously determine lopinavir and ritonavir in human plasma using LC/MS/MS.

It is well known that the combination of high-performance liquid chromatography and mass spectrometry (LC–MS) has a significant impact on drug development over the past decade [4]. Continual improvements in LC–MS interface technologies combined with powerful features for structure analysis, qualitative and quantitative, have resulted in a widened scope of application. These improvements coincided with breakthroughs in combinatorial chemistry, molecular biology, and an overall industry trend of accelerated development [5].

Evidently, LC–MS has become widely accepted as an integral part of the drug development process [6]. This paper describes the simultaneous determination of lopinavir and ritonavir in human plasma using LC/MS/MS. A significant part of the method is the ability to employ parallel HPLC separations with detection by a single MS/MS system to provide the sensitivity and selectivity sufficient to achieve robust analytical results, and high throughput. It has been used to successfully support bioavailability, co-administration of a lopinavir/ritonavir tablet formulation with Efavirenz, and pharmacokinetics studies.

Because a dual system setup (two HPLCs with one MS) was used, the throughput was even doubled compared with the traditional LC/MS/MS system (one HPLC with one MS).

Section snippets

Material and reagents

All aqueous solutions including the HPLC mobile phase were prepared with purified deionized water (Milli-Q, Millipore, France). Glacial acetic acid, sodium carbonate, and ammonium acetate were all ACS grade from J.T. Baker (Phillipsburg, NJ, USA). Hexanes, and methyl acetate were HPLC grade from Sigma–Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was HPLC grade from Aldrich (Milwaukee, WI, USA). Lopinavir, ritonavir, and internal standard (I.S., A-86093) were manufactured and

Results and discussion

This paper describes an analytical method and the validation results for the determination of lopinavir and ritonavir in human plasma using A-86390 as an I.S. A semi-automated liquid–liquid extraction method in 96-well format was used to extract the drug analytes from human plasma samples. An LC/MS/MS method was used to generate the chromatographic peak areas of the analytes and I.S. for quantification.

Since lopinavir has a hydroxyl group and ether bond, and ritonavir has amine groups, both can

Conclusion

A sensitive LC–MS/MS method for simultaneous determination of lopinavir and ritonavir using semi-automatic 96-well liquid–liquid extraction was validated for human plasma. The calibration curves showed goodness of fit over a broad concentration range of 19–5300 ng/mL for lopinavir and 11–3100 ng/mL for ritonavir using a linear regression with 1/x² weighting. This method has successfully supported clinical pivotal bioequivalence, and bioavailablity studies for Kaletra.

Acknowledgement

The authors greatly appreciate Dr. Raymond Wieboldt for his noble help and encouragements. Dr. Wieboldt passed away in 2003.

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