Rapid and Sensitive Method for Quantitative Determination of Lopinavir and Ritonavir in Human Plasma by Liquid Chromatography-Tandem Mass Specrtometry

A rapid and sensitive liquid chromatography-mass spectrometric (LCMS-MS) method for the simultaneous determination of lopinavir and ritonavir in human plasma using abacavir as internal standard has been developed and validated. Sample preparation of plasma involved solid phase extraction. Detection was performed using an Applied Biosystems Sciex API 2000 Mass spectrometer. The assay of lopinavir and ritonavir was linear over the range of 50 ng mL to 20000 ng mL and 20 ng mL to 3000 ng mL respectively with a precision of < 15% and accuracy in the range of 85-115%. The limit of quantification in plasma for lopinavir and ritonavir was 50 ng mL and 20 ng mL respectively. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human plasma.


Chemicals and reagents
Working standard of lopinavir and ritonavir and abacavir were obtained from Cipla.Methanol and acetonitrile of HPLC grade and all other chemicals used were of analytical grade.

Working standard preparation
Stock solutions (1000 µg mL -1 ) of lopinavir, ritonavir and abacavir were prepared in methanol.Working solutions with different concentrations were prepared by dilution of stock with diluent (methanol: water 50: 50).Working solution for internal standard was also prepared in same diluent.

Sample extraction
Plasma samples to be processed were thawed at room temperature.Strata-X 33 µ m Polymeric Reversed Phase (30 mg/1mL) cartridges were conditioned by passing through them 1 mL of methanol followed by 1 mL of water.To prepare samples for loading, 200 µL plasma was mixed with 50 µL of internal standard (approximately 2.000 µg mL -1 ).Sample was vortexed to mix, centrifuged for 5 min at 15000 rpm and loaded on cartridge.Samples were washed with 2 mL water.Analyte and internal standard were eluted from the cartridges using 2000 µ L of mobile phase. 2 µ L of sample was injected onto LC-MS/MS.

Instrumentation and chromatographic conditions
The HPLC system (shimadzu) consist of an isocratic pump, an auto sampler and a Hypersil Hypurity C 18 column (50 x 4.6 mm, id 5 µ ).The mobile phase was prepared by mixing 2% formic acid with acetonitrile in the ratio of 20:80(v/v).Chromatography was performed at ambient temperature with a flow rate of 0.4 mL min -1 .Detection was performed by an Applied Biosystems Sciex API 2000 Mass Spectrometer using atmospheric turbo ion spray for ion production.API was performed in the positive ion mode.The LC-MS-MS detector was operated at unit resolution in the multiple reactionmonitoring (MRM) mode.The transitions of the protonated molecular ions of lopinavir at m/z 629.5 →183.3,ritonavir at m/z 721.4→ 296.3 and abacavir at m/z 287.2→191.2.Product ion mass spectra of lopinavir and ritonavir are shown in Figure 1

Validation procedures Selectivity
Ten different sources of blank plasma samples were extracted and analysed for the assessment of potential interference with endogenous substances.The apparent response at the retention time of lopinavir and ritonavir was compared to the lower limit of quantification.Figure 3

Linearity
Linearity was studied over of 50.000 ng mL -1 to 20000.000ng mL -1 for lopinavir and 20.000 ng mL -1 to 3000.000 ng mL -1 for ritonavir.The calculation was done by linear weighted regression (1/X 2 ).

Accuracy and precision
To validate the method for lopinavir and ritonavir, accuracy and precision were evaluated by analysis of three different concentration levels LQC (i.e.150.0 ng mL -1 of lopinavir and 60.0 ng mL -1 of ritonavir), MQC (i.e.8000.0 ng mL -1 of lopinavir and 800.0 ng mL -1 of ritonavir) and HQC (i.e.18000.0ng mL -1 of lopinavir and 2700.0 ng mL -1 of ritonavir).The results obtained for precision and accuracy of lopinavir and ritonavir are listed in Table 2

Recovery
For the recovery experiment, plasma extracted samples were prepared by spiking both lopinavir and ritonavir at three different concentrations levels (LQC, MQC, HQC) and extracted using extraction procedure.Recovery was carried out by comparing the area obtained from an extracted sample with standard (unextracted) sample.The results obtained for drug and internal standard recovery are listed in Table 2 Stability The stock solution stability at room temperature for 24.0 h was compared with freshly prepared stock solution of lopinavir and ritonavir.Freeze thaw stability was done for LQC and HQC after three cycles at -20 0 C. Room temperature stability in plasma was done for 24 h at two concentrations LQC and HQC.Auto sampler stability was done for 24 h at two concentrations LQC and HQC.Analyte and internal standard responded best to positive ionization using atmospheric turbo ion spray for ion production.In order to get higher response hypersil hypurity C18 (4.6 X 50 mm) column was used.A number of different columns (Nucleosil, Nova pack, hypersil and Symmetry shield) were evaluated and hypersil hypurity C18 was found to give best chromatography with minimal matrix effects.The assay was found to be linear in the concentration range 50.0 to 20000.0 ng mL -1 for lopinavir and 20.0 to 3000.0 ng mL -1 for ritonavir (r > 0.99).Precision and accuracy were satisfactory at the three concentrations.Mean % recovery of the lopinavir and ritonavir was 91.06 and 92.95% respectively.Stability of analyte and internal standard stock solution in methanol was verified on storage for one month at 2-8°C.

Application of method
The method can be successfully applied to determine concentration of drug in a bioequivalence study of lopinavir and ritonavir.

Conclusion
Rapid and sensitive LC-MS-MS method is reported for the simultaneous determination of lopinavir and ritonavir in human plasma.The Assay can be successfully applied to determine concentration of the drug in a bioequivalence study of lopinavir and ritonavir.The method allows high samples throughput due to short run time and relatively simple sample preparation procedure.

Figure 3 .
Figure 3. Representative of plasma blank for lopinavir.

Figure 4 .
Figure 4. Representative chromatogram of plasma spiked with lopinavir at the lower limit of quantification (50.0 ng mL -1 ).

Figure 6 .
Figure 6.Representative chromatogram of plasma spiked with ritonavir at the lower limit of quantification (20.0 ng mL -1 ).

Table 1 .
. Data of precision and accuracy.

Table 2 .
Extraction recovery of drug and internal standard.