1. INTRODUCTION:

Cardiovascular disease, in particular coronary heart disease (CHD), is the principal cause of morbidity and mortality. Elevated plasma total cholesterol and low-density lipoprotein cholesterol levels have been shown repeatedly to be predictive of premature CHD¹. Statins used for reducing serum cholesterol concentrations and lowering the risk of heart attack. Lovastatin hydrolyzes to form a β, Δ-dihydroxy acid, which is an active metabolite, structurally similar to β- hydroxy-β-methylglutaryl coenzymeA (HMG-CoA), a key intermediate in cholesterol biosynthesis. Hydrolyzed lovastatin competes for HMG-CoA reductase, this leads to decrease in cholesterol biosynthesis². Lovastatin and other HMG-CoA reductase inhibitors are also acts as stimulators of bone morphogenetic proteins (BMP) production³.

Plasma levels of lovastatin following therapeutic oral doses are reported to be very low. Probably because only 30% of the dosed lovastatin reaches the systemic circulation and is metabolized to many metabolites⁴, therefore, sensitive and selective methods for the determination of lovastatin have been required for therapeutic drug study.

(a) (b)

Fig. 1. Chemical structure of (a) lovastatin and (b) simvastatin (Internal Standard)

Several papers report gas chromatography coupled to mass spectrometry⁵, reverse phase- high performance liquid chromatography⁶, supercritical fluid chromatography with UV detection⁷ lovastatin in plasma and transient moving chemical reaction boundary method (tMCRBM) in capillary electrophoresis⁸ lovastatin in urine. HPLC method was carried out⁹ using simple protein precipitation for extraction of lovastatin from rat plasma with lower limit of quantitation 60ng/mL

Some of the methods are reported in literature survey such as, determination of lovastatin in human plasma by ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry^10,11, LC-MS/MS method¹² employs solid phase extraction to isolate lovastatin and its hydroxyl acid from mouse and rat plasma. The assay has limit of detection 0.50 ng/mL with overall recovery not more than 55%.

Due to the presence of very low concentration in plasma and liver homogenate, sensitive analytical methods are required for accurate measurement of lovastatin in plasma and liver homogenate. LC-ESI-MS/MS offers a great potential for the analysis of these low levels of lovastatin in plasma and liver homogenate by using the multiple reaction monitoring (MRM) mode of quantitation which makes the analysis highly specific. The application of this assay method to a clinical pharmacokinetic study in healthy male Sprague-Dawley rats following oral as well as intravenous administration of lovastatin is described.

2. MATERIAL AND METHODS:

2.1 Chemicals and reagents

Pure references standard of lovastatin (Fig. 1(a)) and simvastatin (Fig. 1(b)) were obtained from Biocon India LTD. (Delhi, India). LC/MS grade methanol and acetonitrile was obtained by Thomas Baker (Chemical) Pvt. Ltd. (Mumbai, India). Ammonium acetate was procured from Qualligens fine chemicals (Mumbai, India). TDW was prepared in laboratory using distillation assembly. Heparin sodium injection I.P. (Beparine^®, 1000 IU/mL) was procured from Biological Evans Ltd., Hyderabad, India.

Drug-free plasma (Sprague-Dawley male rats) pool was obtained by centrifuging the heparinised blood at 2000 rpm for 10 minutes and separating the supernatant (plasma). Liver homogenate was prepared by homogenization of livers isolated from rats. 20%W/V homogenate was prepared in phosphate buffered saline of pH 7.4. These blank plasma and liver homogenate were stored at -20^οC till use.

2.2 Instrumentation and chromatographic conditions

All analysis was performed using API 4000 LC-MS/MS system (Applied Biosystem, MDS sciex, USA). The system components included Perkin Elmer series 200 quaternary pump, mobile phase vacuum degassing unit, Perkin Elmer series 200 autosampler with peltier temperature set at 4^οC, API 4000 LC-MS/MS detector operated in ESI positive mode. The analysis was performed by Multiple Reactions Monitoring (MRM). Phenomenex ultra sphere Guard (RP-18, 30 x 4.6mm, 5μm) column was used for all chromatographic separations. Mobile phase was 10mM ammonium acetate buffer (pH 4): methanol (10 : 90, v/v). The LC system was operated isocratically at 500 μL/min and at 35^οC.

2.3 Preparation of stock solution of analyte and internal standard

Main stock solution (MSS) of lovastatin (10 μg/mL) was prepared in acetonitrile. Working stock solution-1(WSS-1) was prepared by diluting 20 μL of MSS with 980 μL of acetonitrile to get the concentration 200 ng/mL. Working stock solution-2 (WSS-2) was prepared by diluting 400 μL of MSS with 600 μL of acetonitrile to get the final concentration 4 μg/mL. Standard solution of simvastatin (IS) (100 ng/mL) was prepared in acetonitrile and these stocks were stored at 2–8^οC; they were found to be stable for 30 days.

2.4 Preparation of calibration standards (CS) and quality control (QC) standard

Normal rat plasma and liver were collected from male Sprague-Dawley rats (homogenate using homogenizer) and stored below –20^o C for further use. The calibration and quality control standards were prepared by serial dilution technique by pooling the individually spiked rat plasma and or liver homogenate samples of same concentrations. The individual plasma/liver homogenate standards were prepared by spiking the plasma with working stocks of suitable dilution.

Ten calibration standard (CS) solutions of 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39 and 0.19 ng/mL lovastatin were prepared in acetonitrile. To these solutions fixed volume of IS (10 ng/mL) was added.

Four levels of quality controls (QCs) standard at 100 (HQC), 12.5 (MQC), 1.56 (LQC) and 0.19 ng/mL (LLQC) were prepared. All working solutions were prepared in 2 mL eppendorf tubes.

2.5 Processing of plasma and liver homogenate sample

Sample preparation for calibration standard samples and QC samples involved a simple two-step liquid-liquid extraction (LLE) with ethyl acetate. All plasma/liver homogenate samples were stored at 80º C and thawed at room temperature. The processing volume of plasma/liver homogenate was fixed as 200 μL. The IS solution was spiked to each sample so as to get a final concentration of 10 ng/mL. The samples were vortex mixed prior to addition of the extraction solvent. To each aliquot 2.0 mL ethyl acetate was added and vortex mixed for two minutes and centrifuged at 2000 rpm for 5 min in order to separate two immiscible layers. Then after snap freezing, the upper organic layer was transferred to another set of clean glass tubes and evaporated in Savant Speed Vac (USA) vacuum evaporator. This extraction procedure was repeated one more time. The dry residues were combined and reconstituted in 200 μL of acetonitrile and injected to HPLC-MS-MS.

2.6 Optimization of method

Triple quadrapole mass spectrometer was operated using a standard ESI source in positive ion mode. Data acquisition and analysis were performed using Analyst software (version 1.4). Nitrogen was used as both nebulizing gas (30 L/h) and as drying gas (40 L/h). The source of temperature was set at 400ºC, LC/SIR (selective ion recording)/MS optimization was performed by constant infusion of the mixture of analytes using Harvard infusion pump, USA. The cone voltage, capillary voltage, nebulizing and dry gas condition were kept at default values. The dwell time was 200 msec, SIR masses used for quantification were m/z 422.3 (M+NH₄) and 436.5 (M+NH₄) for lovastatin and simvastatin respectively. Collision energies (CE) for the fragmentation of precursor to product ions were optimized by constant infusion. Each analyte was optimized to obtain the most intense precursor to product ion transitions. The CEs for 422.3 – 285.1 transition was set at 18KV for lovastatin (Fig. 2) and for 436.5 – 199.2 transition was set at 20 KV for simvastatin (Fig. 3).

2.7 Method Validation

The method validation of lovastatin in rat plasma and liver homogenate were performed according to the FDA guidelines¹³. The parameters determined were speciﬁcity and sensitivity, linearity, precision and accuracy, extraction recovery, matrix effect, and stability. Specificity was assessed by comparing the chromatograms of six different batches of blank plasma and liver homogenate and analyzed to determine the extent of interference by endogenous plasma components at the retention time of both analyte and IS. Sensitivity was determined by analyzing six replicates of plasma and liver homogenate samples spiked with the lowest level of the quality control standard.

Linearity was evaluated using plasma and liver homogenate samples spiked with lovastatin at concentration ranges 0.19-100 ng/mL. The IS solution was spiked to each sample so as to get a final concentration of 10 ng/mL The calibration curves were drawn by plotting area ratios of analyte against the concentration of each calibration standard.

Intra-day precision and accuracy were determined by analyzing six replicates at four different QC levels on same day. Inter-day precision and accuracy were determined by analyzing six replicates at four different QC levels on ﬁve different days.

The absolute recoveries (extraction efficiency) of lovastatin were determined at three different concentrations 1.56, 12.5 and 100 ng/mL (low, middle and high concentrations) by standard addition method. A known amount of lovastatin was added to rat plasma and liver homogenate prior to extraction and IS was added after extraction to eliminate bias which may introduced by sample processing. The matrix effect was assessed as a percentage of the peak areas of control plasma extracted and then spiked with analyte, to neat standards injected directly in the same reconstitution solvent. The matrix effect was carried out on six different lots of blank plasma and liver homogenate and at low, middle and high QC levels.

2.8 Stability

Stability tests were conducted to evaluate the analyte stability in stock solutions and in plasma as well as liver homogenate samples under different conditions. The stock solution stability at room temperature and refrigerated conditions (2–8ºC) was performed by comparing the area response of the analytes (stability samples) with the response of the sample prepared from fresh stock solution.

2.8.1 Bench top stability

Six aliquots of unextracted QC samples at low, middle and high concentration were kept at ambient temperature (25ºC) for 8 h in order to determine bench top stability of lovastatin in plasma and liver homogenate. Then, the samples were processed and analyzed and the concentrations obtained were compared with the nominal values.

2.8.2. Freeze and thaw stability

The effect of freeze and thaw cycles on the stability of plasma and liver homogenate containing lovastatin was determined by subjecting six aliquots of unextracted QC samples at low, middle and high to five freeze–thaw cycles. After completion of every cycle, the samples were analyzed and the experimental concentrations were compared with the nominal values. The accuracy values of three concentrations in four freeze–thaw cycles were calculated.

2.8.3. Short-term stability

Six aliquots of unextracted QC samples at low, middle and high concentration were kept at ambient temperature (25ºC) for 24 h in order to determine the short-term stability of lovastatin in plasma and liver homogenate. Then, the samples were processed and analyzed and the concentrations obtained were compared with the nominal values.

2.8.4. Long-term stability

Six aliquots of unextracted QC samples at low, middle and high concentration were stored at -70ºC for 30 days. Then, the samples were processed and analyzed and the concentrations obtained were compared with the nominal values

Samples were considered to be stable if assay values were within the acceptable limits of accuracy (85–115%) and precision (≤ 15% RSD).

2.8.5. Post-preparation stability

In order to estimate the stability of lovastatin in the prepared sample, six aliquots of QC samples at low, middle and high concentration were kept in an auto sampler maintained at 4ºC for about 4 h. Then, the samples were analyzed and the concentrations obtained were compared with the nominal values.

2.9 Pharmacokinetic study

Male Sprague-Dawley rats weighing 250±10 g were administered as a single dose orally (20mg/kg) and intravenously (1mg/kg) of lovastatin through tail vein injection. The blood samples were collected by cardiac puncture at 0.1, 0.5, 1, 2, 4, 8, 12 and 24 h time points by sparse sampling method using 3 rats for each time interval. The blood samples were collected in heparinized glass tubes and immediately centrifuged (2000 rpm for 10 min) to separate plasma. Collected plasma was stored at 80ºC until analysis. The concentrations of lovastatin in these plasma samples were determined using the procedure described in section 2.5. The determined concentrations were subjected to non compartmental analysis in WinNonlin software (version 1.5) to calculate pharmacokinetic parameters.

3 RESULTS AND DISCUSSION:

3.1 Sample preparation and optimization of LC/MS/MS

The use of a short chromatography column Phenomenex ultra sphere Guard (RP-18, 30 x 4.6mm, 5μm) helped in elution of lovastatin in a very short time. It gave a symmetric peak shape for all analytes with run time less than 2 min.

MS parameters were optimized by infusing the standard analyte solution into the mass spectrometer using electro-spray as the ionization source and operating in the MRM mode. The signal intensities obtained in positive mode were much higher than those in negative ion mode since the analyte and IS have the ability to accept protons. The most sensitive mass transition was monitored at m/z 422.3 (M+NH4) and 436.5 (M+NH4) for lovastatin and simvastatin respectively. Each analyte was optimized to obtain the most intense precursor to product ion transitions. In order to minimize undesirable fragmentation and achieve highest response, various fragmentor voltages were tested. As LC-MRM provides sensitivity and selectivity requirements for analytical methods, it is proved to be a very powerful technique for pharmacokinetic studies¹⁴.

3.2 Specificity and sensitivity (LLOQ)

Rat plasma and rat liver homogenate tested for the presence of endogenous substances that might interfere at the retention times of peaks of interest. No signiﬁcant direct interference in the blank plasma traces was observed from endogenous substances in drug-free plasma and liver homogenate at the retention time of the analytes.

The lowest quantification limit of an analyte was tested at different levels ranging from 0.02-1.56 ng/mL and 0.19 ng/mL was found as LLOQ for lovastatin with acceptable accuracy and precision.

3.3. Linearity, precision and accuracy

Calibration curves were plotted as the peak area ratio (drug/IS) vs. drug concentration. Ten point calibration curves (n=6) were found to be linear over the concentration range of 0.19–100 ng/mL. The mean correlation coefficient of the calibration curves generated was ≥ 0.99 both for plasma and liver homogenate (Fig. 4, Table 1)

Table 1 Statistical evaluation of calibration data of lovastatin

Parameters	Rat Plasma	Liver Homogenate
Linearity range (ng/mL)	0.19–100	0.19–100
Slope	0.000797	0.00595
Intercept	0.00373	0.000415
Correlation coefficient (r)	0.9995	0.9951
LOD (ng/mL)	0.02	0.02
LOQ (ng/mL)	0.19	0.19

Accuracy and precision for the assay were determined by calculating the intra-day and inter-day variation at four concentrations 0.19, 1.52, 12.5 and 100 ng/mL in six replicates. As shown in Table 2.

Table 2 Intra-day and inter-day precision and accuracy of lovastatin

QC	QC (Spiked concentration ng/mL)	Intra-day (n=6)			Inter-day (n=6)
QC	QC (Spiked concentration ng/mL)	Mean concentration found (ng/mL)	Accuracy (mean±SD^a)	Precision (% RSD)^b	Mean concentration found (ng/mL)	Accuracy (mean±SD^a)	Precision (% RSD)^b
Rat Plasma
LLQC	0.19	0.18	94.84 ± 6.03	6.89	0.17	90.22 ± 8.35	8.95
LQC	1.56	1.49	95.68 ± 4.37	4.22	1.61	103.35 ± 5.87	6.12
MQC	12.50	11.98	95.90 ± 5.01	4.96	11.16	96.02 ± 2.44	2.42
HQC	100.00	96.68	96.68 ± 5.39	5.42	101.26	101.26 ± 3.58	3.02
Liver homogenate
LLQC	0.19	0.19	99.16 ± 4.14	4.12	0.18	95.12 ± 7.69	7.58
LQC	1.56	1.47	94.31 ± 3.33	3.48	1.66	106.36 ± 7.01	6.69
MQC	12.50	12.24	98.01 ± 2.89	2.77	11.96	95.91 ± 3.56	3.62
HQC	100.00	92.36	92.36 ± 3.38	3.30	93.28	93.28 ± 6.29	6.22

^aSD, standard deviation

^bRSD, relative standard deviation

Table 3 Recovery and matrix effect study of lovastatin (n=6)

QC	Added Concentration (ng)	Rat Plasma		Liver homogenate		Absolute matrix effect (Mean ± SD^a)
QC	Added Concentration (ng)	Measured Concentration (ng)	% Recovery (Mean ± SD^a)	Measured Concentration (ng)	% Recovery (Mean ± SD^a)	Absolute matrix effect (Mean ± SD^a)
LQC	1.56	1.47	94.53 ± 5.28	1.51	96.82 ± 9.22	96.29 ± 4.58
MQC	12.50	11.98	96.12 ± 5.51	11.72	93.79 ± 3.59	92.36 ± 4.21
HQC	100.00	93.26	93.26 ± 4.39	90.71	90.71 ± 5.28	95.44 ± 2.94

^aSD, standard deviation

Table 4 Stability of lovastatin in rat plasma and liver homogenate at three QC levels (n = 6)

Stability	Rat plasma			Liver homogenate
	% Accuracy (mean ± SD)			% Accuracy (mean ± SD)
	LQC 1.56 (ng/ml)	MQC 12.5 (ng/ml)	HQC 100 (ng/ml)	LQC 1.56 (ng/ml)	MQC 12.5 (ng/ml)	HQC 100 (ng/ml)
Bench top stability (at 25ºC for 8h)	100.31 ± 5.73	99.39 ± 1.26	100.13 ± 5.71	99.88 ± 8.29	97.38 ± 5.24	98.63 ± 2.22
Freeze-haw stability (five Freeze-haw cycles)	98.44 ± 4.33	99.17 ± 7.11	98.91 ± 2.56	99.25 ± 6.62	100.31 ± 4.98	99.73 ± 3.41
Short-term stability (at 25ºC for 24h)	98.66 ± 4.78	100.84 ± 4.48	98.62 ± 2.21	100.29 ± 1.28	100.62 ± 5.70	100.67 ± 1.05
Long-term stability (at -70ºC for 30 days)	97.56 ± 3.31	98.24 ± 3.26	99.22 ± 1.26	97.31 ± 3.84	98.67 ± 3.29	97.19 ± 1.68
Post-preparation stability (at 4ºC for 4h)	102.33 ± 4.23	101.41 ± 3.59	99.66 ± 4.58	98.34 ± 2.45	98.22 ± 3.21	100.89 ± 3.01

SD, standard deviation

The precision and accuracy of the present method conform to the criteria for the analysis of biological samples according to the guidance of USFDA ¹⁵. Deviation values for the intra-day and inter-day precision and accuracy were all within 15% of the relative standard deviation (RSD) at low, middle and high quality control levels, whereas within 20% at LLOQ quality control level. These results indicate that the method was reliable within the analytical ranges and the use of the internal standard was very effective for reproducibility by LC-MS/MS.

3.4. Extraction recovery and matrix effect

The extraction mean recoveries of lovastatin from rat plasma and liver homogenate were >90%. In terms of matrix effect, the percentage of the peak areas were between 85 and 115%, which means no matrix effect for lovastatin in this method (Table 3)

3.5. Stability

The stock solutions of lovastatin in plasma and liver homogenate were found to be stable at room temperature for 8 h, 24h, at freeze and thaw stability, at the -70ºC for 30 days and at 4ºC for 4 h, (Table 4). The stock solutions were stable for at least 30 days. The difference values between the fresh samples and the test solution in stock solution stability were <5% for lovastatin. The results from all stability tests presented in demonstrated a good stability of lovastatin at over all steps of the determination.

3.6. Pharmacokinetic study

This analytical method was used to calculate PK parameters after administration of single dose of lovastatin by oral as well as intravenous (iv) route to male Sprague-Dawley rats. Lovastatin is rapidly absorbed after oral administration and peak plasma concentration is achieved within 2 h. Afterwards it is rapidly eliminated from plasma which is also evident from iv profile. After iv administration, lovastatin levels were not detectable beyond 12 h. The absolute bioavailability of lovastatin in our experiment was found to be 9.6%. MRT and t_1/2by both the routes were comparable. The PK parameters indicate that drug remains in plasma for a short time and is widely distributed to other compartments. The calculated PK parameters like AUC are in accordance with previous report¹⁶. The differences in some of the parameters can be accounted for the difference in dosing formulation and the doses administered (Fig. 5(a and b), Table 5).

Table 5 Pharmacokinetics parameters of lovastatin (n = 6, Mean ± SD)

PK Parameter	Oral	Intravenous
C_max (ng/mL)	40.73	---
T_max (h)	2	---
Elimination t_1/2 (h)	5.76	4.65
AUC_(0-24) (ng.h/mL)	176.6	91.84
V_d (L/kg)	85.96	56.78
Cl (L/h/kg)	9.91	8.45
MRT (h)	7.96	6.59
Absolute bioavailability (%)	9.6

4 CONCLUSION AND PERSPECTIVES:

A rapid, sensitive and reproducible high-performance liquid chromatography-electrospray tandem mass spectrometry method was described for quantitation of lovastatin in rat plasma and rat liver homogenate. To the best of knowledge, there was no published method so far for determination of lovastatin in rat plasma and liver homogenate. The proposed developed method shows significant advantages in comparison with several reported methods, as it offering superior sensitivity with LLOQ of 0.19 ng/mL and desired precision and accuracy in total run time of less than 2.0 min. From the results of all the validation parameters, we can conclude that the developed method can be useful for bioavailability/bioequivalence study. The method has been successfully applied to pharmacokinetic study.

5. ACKNOWLEDGMENT:

The authors gratefully acknowledge Head of the Department, Department of Pharmaceutical Sciences; RTM Nagpur University, Nagpur for providing necessary facilities for carrying out this study.

6. REFERENCES:

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3 Mundy G, et al. Stimulation of bone formation in-vitro and in rodents by statins. Science. 286 (5446);1999: 1946-1949.

4 Tang BK and Kalow W. Variable action of lovastatin by hydrolytic enzymes in human plasma and liver. European Journal of Clinical Pharmacology. 47 (5); 1995: 449-451.

5 Morris MJ, et al. Determination of the HMG-Co-A reductase inhibitors simvastatin, lovastatin, and pravastatin in plasma by gas chromatography/chemical ionization mass spectrometry. Biological Mass Spectrometry. 22 (1); 1993:1-8.

6 Ye LY, et al. Determination of lovastatin in human plasma using reverse phase-high performance liquid chromatography with UV detection. Therapeutic Drug Monitoring. 22 (6); 2000: 737-741.

7 Strode JT, et al. Feasibility of lovastatin analysis by packed column supercritical fluid chromatography with ultraviolet detection. Journal of Pharmaceutical and Biomedical Analysis. 20 (1-2); 1999: 137-143.

8 Li M, et al. Stacking and quantitative analysis of lovastatin in urine samples by the transient moving chemical reaction boundary method in capillary electrophoresis. Analytical and Bioanalytical Chemistry. 387 (8); 2007: 2719-2725.

9. Zhang Z and Yang Z. HPLC determination of lovastatin in rat tissue. Chromatographia. 66 (7-8); 2007: 487-491.

10 Yuan H, et al. Determination of lovastatin in human plasma by ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry and its application in a pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis. 46 (4); 2008: 808-813.

11 Wang D, et al. Determination of lovastatin in human plasma by ultra-performance liquid chromatography/ electrospray ionization tandem mass spectrometry. Biomedical Chromatography. 22 (5); 2008: 511-518.

12 Wu Y, et al. Microsample determination of lovastatin and its hydroxy acid metabolite in mouse and rat plasma by liquid chromatography/ionspray tandem mass spectrometry. Journal of Mass Spectrometry. 32 (4); 1997: 379-387.

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14 Polangani SR, et al. Simultaneous determination of atorvastatin, metformin and glimepiride in human plasma by LC–MS/MS and its application to a human pharmacokinetic study. Journal of Pharmaceutical Analysis. 3 (1); 2013: 9-19.

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Received on 28.08.2014 Modified on 09.09.2014

Asian J. Research Chem. 7(10): October- 2014; Page 870-876

*Corresponding Author E-mail: sanjuwalode@rediffmail.com, sgwalode.sips@sinhgad.edu

1. INTRODUCTION:

5 Morris MJ, et al. Determination of the HMG-Co-A reductase inhibitors simvastatin, lovastatin, and pravastatin in plasma by gas chromatography/chemical ionization mass spectrometry. Biological Mass Spectrometry. 22 (1); 1993:1-8.

8 Li M, et al. Stacking and quantitative analysis of lovastatin in urine samples by the transient moving chemical reaction boundary method in capillary electrophoresis. Analytical and Bioanalytical Chemistry. 387 (8); 2007: 2719-2725.

9. Zhang Z and Yang Z. HPLC determination of lovastatin in rat tissue. Chromatographia. 66 (7-8); 2007: 487-491.