Estimation of Isradipine in Human Plasma by LCMS/MS

 

Anil Kumar Meher,  Anandan P and Arindam Mukhopadhyay*

Bioanalytical Laboratories, Lotus Clinical Research Academy Pvt. Ltd., 582, KCA Enclave, Koramangala        8th Block, Bangalore – 560 095

*Corresponding Author E-mail: arindam@lotusacademy.co.in

 

ABSTRACT:

A rapid and sensitive method for quantification of the antihypertensive drug, isradipine in human plasma using LCMS/MS has been developed. Protein was precipitated from the plasma by 20% trichloroacetic acid. It was then made alkaline with 1M sodium hydroxide. Isradipine and the internal standard (IS), Nimodipine, were extracted by a simple liquid –liquid extraction method using extraction solvent mixture (n-Hexane: t-butyl methyl ether- 70:30, v/v). The extract was then separated by a reverse phase HPLC  on a XTerra MSC18 column (100mm x 3.0 mm, 5µm) using solvent mixture 42:58, v/v ( Mobile phase 1: 0.1%, v/v, Formic Acid; Mobile phase 2: Acetonitrile: Methanol- 75:25, v/v). Flow rate was 0.5 ml/min without splitter and the column oven temperature was 35ºC. The analytes were then detected by monitoring the transitions m/z 372.1        m/z 312.0 for isradipine and 419.0        343.0 for nimodipine using API 3000 LCMS/MS system (Applied Biosystems) with Turbo Ion Spray interface. A good linear standard curve was obtained within the range of 0.051ng/ml – 20.448ng/ml (r > 0.9973). The lower limit of quantitation (LLOQ) was 0.051 ng/ml. The overall accuracy and precision were 94.28% and 6.19%, respectively. No significant degradation for isradipine in human plasma was observed at room temperature (7h) or when subjected to freeze thaw procedures (3 cycles). No significant metabolic compounds were found to interfere with the analysis. The results of method validation offer the acceptable performance for the specificity, selectivity, sensitivity, linearity, accuracy, precision and stability. This method can be applied for quantitation of isradipine in dosed human plasma samples.

 

KEYWORDS: Isradipine, Nimodipine, LCMS/MS, Validation, human plasma.

 


 

INTRODUCTION:

The calcium channel  blocker of the dihydropyridine class, Isradipine (IUPAC name: 3-methyl 5-propan-2-yl 4-(2,1,3-benzoxadiazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate ; tradenames DynaCirc, Prescal) is usually prescribed for the treatment of high blood pressure in order to reduce the risk of stroke and heart attack1,2. More recent research in animal models suggests that isradipine may have potential uses for treating Parkinson’s disease3.

Although a number of publications is available on action of isradipine, only little information is available on its estimation in biological matrix 4 - 9. This paper is reporting a simple, accurate, sensitive and rapid method for quantification of isradipine in human plasma developed using LCMS/MS which has been validated in accordance to the published FDA guidelines.

 

EXPERIMENTAL:

Materials and Reagents:

Acetonitrile, methanol, Tertiary butyl methyl ether (TBME) (HPLC grade) were purchased from Spectrochem whereas n-hexane (HPLC grade) was from Merck. Other reagents used were of analytical grade.  Millipore (USA) deionized water was used throughout the procedure.

 

Human plasma containing K2 EDTA as an anti-coagulant was procured from Lotus Labs, Bangalore.

Isradipine and nimodipine (internal standard) were from Varda Scientific, Ahmedabad, India. Stock solution (1mg/ml) of nimodipine was prepared in methanol. Final concentration is made using the potency and actual amount weighed. Serial dilutions were made using methanol: water (1:1, v/v) to obtain a final concentration 100ng /ml.

 

Similar to nimodipine, stock solution (1mg/ml) of isradipine was prepared in methanol and final concentration was corrected based on its potency and actual amount weighed. Stock solution was serially diluted with methanol: water (1:1, v/v) to obtain a concentration range of 3ng /ml to 10000ng /ml.

Quality control (QC) samples for analyte were prepared in the range of 0.050 – 15.6 ng /ml using K2 - EDTA plasma.

Construction of Calibration Plot:

The blank plasma was spiked with isradipine to obtain the final concentration 0.050 – 20.6 ng /ml. Final concentration was again corrected based on potency and stock weight. 50µl of internal standard (nimodipine, 100ng/ml) was added to all these isradipine spiked plasma (600µl). 50µl of 20% trichloroacatic acid was added to the mixture and vortexed for 30 sec. It was followed by addition of 0.6ml of 1M NaoH and vortexed again. Isradipine was extracted from the plasma by liquid –liquid extraction after addition of 2.5ml of extraction solvent mixture (n-Hexane: Tertiary butyl methyl ether :: 70:30v/v). The RIA vials were then shaken on the vibramax at 2500 rpm for 10 min. The organic layer was separated by centrifuging it at 4500 rpm for 10 min at 40C. Top organic layer (2ml) was transferred to a new RIA vial, evaporated to dryness with a stream of nitrogen gas at 250C for 15 min. It was then reconstituted with 0.3ml of mobile phase [0.1% Formic Acid : Organic Mixture (Acetonotrile: Methanol,75:25) :: 42:58v/v].  20µl was injected to LCMS/MS for analysis.

 

Quality control (QC) samples, marked as LLOQC, LQC, MQC and HQC, containing 0.050 ng /ml, 0.131 ng /ml, 6.240 ng /ml and 15.6 ng /ml of the drug respectively, were prepared in blank plasma.

 

Sample Analysis:

Isradipine was isolated from plasma by liquid – liquid extraction followed by evaporation and reconstitution as mentioned above.

 

Chromatography:

The drug was separated on a XTerra MSC18 (100mm x 3.0 mm, 5µm) (Waters) using the binary pump [pump A - 0.1% formic acid: pump B – Organic Mixture (Acetonotrile: Methanol, 75:25, v/v):: 42:58, v/v] at a flow rate of 0.5 ml/min in Shimadzu UFLC Prominence attached to API 3000 Mass spectrometer (Applied Biosystems, USA) with an ESI interface. The column oven temperature was maintained at 350C and the run time was 5.0 min. The analytes were detected on mass spectrometer operating in the positive electrospray ionization mode with multiple reactions monitoring (MRM). The MRM transitions monitored were m/z for parent ion 372.10 and daughter ion 312.0 (isradipine), m/z for parent ion 419.0 and daughter ion 343.0 (nimodipine). Data were acquired and processed with Analyst software 1.4.1.

 

RESULTS AND DISCUSSION:

Specificity and Selectivity:

The retention times for isradipine and the internal standard were 3.42 and 3.97 minutes, respectively (Fig.1).

 

The procedure is specific to isradipine as there was no interfering peaks observed in the blank at the retention times corresponding to drug and I.S (Table 1). Similarly, no matrix effect was found while analyzing the human plasma samples, calibration standards and QC samples (Table 2).

 

Linearity of the Calibration Plot

A linear calibration plot of isradipine and I.S. in the concentration ranges of 0.051 to 20.448 ng/ml with correlation coefficient > 0.9973 was achieved.

 

The accuracy and precision were assessed by the repeated analysis of plasma samples containing different concentrations of Isradipine on three different days. A single run consists of Aqueous standard, reagent blank, zero sample calibration standard plus 6 replicates of the LOQQC, LQC, MQC and HQC samples. Precision for LOQQC was found to be 12.18 % and for LQC, MQC, HQC was found to 4.88 % to 6.30 %. The percentage of nominal concentration ranged 92.79 % to 95.84 % for LQC, MQC and HQC and 89.54 % for LOQQC (Table 3).

 

Recovery:

To determine absolute recovery percentage, the peak area of isradipine obtained by injecting 6 extracted samples of LQC, MQC and HQC sample was compared with the peak obtained by injection of standard solutions of the same concentration. Average recovery of isradipine from biological matrix was 72.1 %. (Table 4).

 

Stability:

Short – Term/bench - top stability:

In order to determine the stability of the sample during analysis, six aliquots of LQC and HQC samples were thawed and kept at room temperature for 7 hours, which has been decided based on the time required for analysis. The samples were then processed and analyzed as mentioned above. When these results were compared with those obtained from the freshly spiked samples, no significant differences were noticed indicating that the analyte was stable at room temperature (Table 5).

 

 


Table 1:  Selectivity for Isradipine

K2EDTA Plasma Lot No.

Analyte Area

IS Area

Area Ratio

Avg. Area Ratio + SD

Analyte Signal to Noise Ratio

Avg Analyte Signal to Noise Ratio + SD

1

3360

807445

0.0042

0.00406 +

0.000242

 

77.852

69.635 + 10.68

 

2

3320

783132

0.0042

70.896

3

2811

777448

0.0036

54.366

4

3173

787068

0.004

58.501

5

3258

771055

0.0042

78.913

6

3575

853453

0.0042

77.283

 

Table 2: Matrix Effect for Isradipine

Aqueous LQC Analyte

(Area count) + SD (n=6)

Post spiked LQC analyte

(Area count ) + SD (n=6)

Percentage matrix Effect + SD (n=6)

11131 + 435.48

10895.5 + 164.12

97.88 + 1.47

 

 

Table 3 : Inter day Precision and Accuracy Batch for Isradipine

Actual Conc. of LOQQC (ng/ml)

Avg. Conc + SD (n=24) LOQQC (ng/ml)

Actual Conc. of LQC ng/ml)

Avg. Conc + SD (n=24)

LQC (ng/ml)

Actual Conc. of MQC (ng/ml)

Avg. Conc + SD (n=24)

MQC (ng/ml)

Actual Conc. of HQC(ng/ml)

Avg. Conc + SD (n=24)

HQC (ng/ml)

0.051

0.046 + 0.006

0.135

0.129 +  0.008

6.144

5.880 +  0.359

15.359

14.252 + o.695

 

 

 

Table 4:  Recovery of Analyte from Biological Matrix (Isradipine)

Mean Area of Unextracted LQC (n=6)

Mean Area of Extracted LQC (n=6)

Mean % Recovery after volume correction

(LQC)

Mean Area of Unextracted MQC (n=6)

Mean Area of Extracted MQC (n=6)

Mean % Recovery

after volume correction (MQC)

Mean Area of Unextracted HQC (n=6)

Mean Area of Extracted HQC (n=6)

Mean % Recovery

after volume correction (HQC)

Avg. % Recovery

12409

7778

78.35

531132

293036

68.96

1369526

756968

69.09

72.13

 

 


 

Table 5: Bench Top Stability of Analyte in Biological Matrix for Isradipine

Comparison of QC samples

LQC

(Actual Conc. 0.135 ng/ml)

HQC

(Actual Conc. 15.359 ng/ml)

Fresh Sample

After 7 hours (ng/ml)

Fresh Sample

After 7 hours (ng/ml)

0.130 + 0.010

0.137 + 0.010

14.154 + 0.390

14.303 + 0.393

 

Auto sampler stability:

The stability of the processed samples in the auto sampler during analysis was determined by using six aliquots of LQC, MQC and HQC samples. The stability of drug and IS were assessed for 20 hours, the expected run time for batches of validation samples. The results were then compared with that of freshly spiked samples. No significant difference in the results indicated that the isradipine and IS are stable for at least 20 hour in the auto sampler (Table 6).

 

Table 6: Stability of Isradipine in Auto Sampler at 10o C

Comparison of QC samples

LQC

(Actual Conc. 0.135 ng/ml)

HQC

(Actual Conc. 15.359 ng/ml)

Fresh Sample

After 20 hrs (ng/ml)

Fresh Sample

After 20 hrs (ng/ml)

0.130 + 0.010

0.135 + 0.007

14.154 + 0.390

14.406 + 0.274

 

Freeze – Thaw stability:

Analyte stability was determined after three freeze – thaw cycles for six aliquots of each of the LQC and HQC. The samples were stored below – 200C for 24h and then allowed to thaw at room temperature unassisted. After complete thawing, the samples were stored at same temperature for 12h. The freeze – thaw cycle was repeated twice before analyzing the samples. Comparison of the results with the fresh QC samples indicated no differences (Table 7).

 

 

Table 7: Stability of Isradipine in Biological Matrix after three Freeze Thaw Cycles Below -20ºC

Comparison of QC samples

LQC

(Actual Conc. 0.135 ng/ml)

HQC

(Actual Conc. 15.359 ng/ml)

Fresh Sample

After 3 FT Cycles (ng/ml)

Fresh Sample

After 3 FT Cycles (ng/ml)

0.130 + 0.010

0.131 + 0.007

14.154 + 0.390

13.914 + 0.318

 

CONCLUSION:

This LCMS/MS method for estimation of isradipine is accurate, precise, linear, rugged, robust, simple and rapid. The method is suitable for pharmacokinetic studies of isradipine in human plasma as per the regulatory authorities and may also be used for quality control of raw materials, formulations and dissolution studies.

 

REFERENCES:

1.       Hattori T, Wang P (2006). "Calcium antagonist isradipine-induced calcium influx through nonselective cation channels in human gingival fibroblasts.". Eur J Med Res 11 (3): 93–6

2.       Ganz M, Mokabberi R, Sica D (2005). "Comparison of blood pressure control with amlodipine and controlled-release isradipine: an open-label, drug substitution study.". J Clin Hypertens (Greenwich) 7 (4 Suppl 1): 27–31.

3.       Chan CS, Guzman JN, Ilijic E, Mercer JN, Rick C, Tkatch T, Meredith GE, Surmeier DJ (2007). "'Rejuvenation' protects neurons in mouse models of Parkinson's disease.". Nature 447 (3): 1081–1086.

4.       Kalpana Barhwal, Sunil Kumar Hota, Iswar Baitharu, Dipti Prasad, Shashi Bala Singh and G. Ilavazhagan Isradipine antagonizes hypobaric hypoxia induced CA1 damage and memory impairment: Complementary roles of L-type calcium channel and NMDA receptors Neurobiology of Disease, Volume 34, Issue 2, May 2009, Pages 230-244

5.       RM Aramisova, AA El'garov Isradipine in arterial hypertension in motor vehicle drivers Med Tr Prom Ekol (1996) 0: 21-3

6.       Pinquier JL, Urien S, Chaumet-Riffaud P, Comte A, Tillement JP  Binding of [3H]isradipine (PN 200-110) on smooth muscle cell membranes from different bovine arteries J Cardiovasc Pharmacol (1988) 11: 402-6.

7.       K. Schönholzer and C. Marone  Pharmacokinetics and dialysability of isradipine in chronic haemodialysis patients  European Journal of Clinical Pharmacology, Volume 42, Number 2, 231-233, 1992

8.       F Cachat and A Tufro Phenytoin/isradipine interaction causing severe neurologic toxicity The Annals of Pharmacotherapy: Vol. 36, No. 9, pp. 1399-1402.  2002.

9.       Al-Suwayeh, Saleh A  Quick, simple and sensitive HPLC method for determination of isradipine in plasma and its application in pharmacokinetic studies Analytical Letters 35, 1205 – 1213, 2002

 

 

 

Received on 27.01.2011        Modified on 22.02.2011

Accepted on 07.03.2011        © AJRC All right reserved

Asian J. Research Chem. 4(5): May, 2011; Page 750-753