A Validated Reverse Phase LC-MS Method for quantification of drotaverine in Biological matrices

 

K. Naga Prashant, A. Vijaya Lakshmi*

Department of Pharmacognosy, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies, Pallavaram, Chennai – 600117, Tamil Nadu, India.

 

ABSTRACT:

A simple and precise liquid chromatography coupled with mass spectrometry LC-MS method was developed for determination and quantification of Drotaverine in human plasma and validated. The drug was separated by using liquid – liquid extraction (LLE) Method. A C18 column was employed for the separation of the drug on a gradient mode by using high-performance liquid chromatography (HPLC). The lower limit of quantification (LLOQ) of Drotaverine was 1.013ng/mL. The calibration curve ranges from of 0.993ng/mL to 498.245ng/mL with desirable linearity and r2 greater than 0.99. The intra-day accuracy was within 100.583–98.033% with coefficient of variation 0.90–0.14%. No significant interference observed by endogenous peak at the retention time of Drotaverine and IS. The assay was free from any matrix effect, precise recovery across the calibration curve range and samples were stable under all experimental conditions.

 

 

 

INTRODUCTION:

Drotaverine chemically is a benzylisoquinoline derivative which is very similar to that of papaverine with empirical formula C24H31NO4 and molecular weight of 397.50. The structure of the drotaverine is shown in figure no. 1. It is an antispasmodic agent^[1] which acts by inhibiting phosphodiesterase IV enzyme, specially used for treating smooth muscle spasm and pain. It is also widely used to reduce cervical spasm and excessive labor pain^[2]. It is used to relieve pain caused due to headache, irritable bowel syndrome^[3] and also during menstrual periods. It is official in Martindale, The Extra Pharmacopoeia^[4]. Literature reveled that several methods have been reported for quantitative estimation of drotaverine hydrochloride using UV spectrophotometry^[5,6,7] HPLC^[8,9,10] thin layer chromatography^[11,12] and voltammetry^[13].

 

Fig. No: 1 Structure of Drotaverine

 

The aim of present study was to develop a simple, rapid, sensitive and precise method of quantification by using liquid chromatography coupled with mass spectrometry (LC/MS).

 

EXPERIMENTAL:

Chemicals and materials:

Reference standards of Drotaverine (mol.wt. 397.50; 99.3% w/w), internal standard (IS) and Control buffered potassium salt of ethylene di-amine tetra acetic acid (K EDTA) human plasma stored at –70°C until use were provided by K P Labs, Hyderabad. The structure of Drotaverine has been shown in Figure 1. All other reagents/chemicals were of analytical reagent grade.

 

LC-MS/MS instrumentation and conditions:

A HPLC system (Shimadzu) with Atlantis T3 column was employed in this study. The column oven and auto-sampler temperature were maintained at 40±2°C and also the flow rate was set at 1.000mL/min. Ionization and detection of analyte and ion spray (IS) was performed on a triple quadrupole mass spectrometer, application programming interface 4000 LC-MS equipped with turbo IS, operated in the positive ion mode. Quantitation was performed using the multiple reaction monitoring (MRM) mode to monitor protonated precursor to product ion transition of m/z 100 → 500 amu. All the parameters of HPLC and MS were controlled by analyst software version 1.4.2.

 

The source dependent parameters maintained for analyte and IS were GS1: 50.00 psi, GS2: 50.00 psi, IS voltage: 2000.00 V, turbo heater temperature (TEM): 600.00°C, collision activation dissociation (CAD): 6.00 psi, curtain gas (CUR): 25.00 psi. The compound dependent parameters like declustering potential (DP) were optimized at 55.00 V, collision energy (CE) was 27.00 V, cell exit potential (CXP) was kept at 6.00 V and entrance potential (EP) was 10.00 V.

 

Preparation of calibration and quality control samples:

Preparation of standard stock and plasma samples:

The standard stock solution of Drotaverine (1mg/10mL) is prepared by dissolving in the requisite amount of methanol. Further dilutions from the stock solutions were prepared using the diluent solution (methanol: Milli-Q water: 50:50, v/v) for spiking in plasma to obtain calibration curve (calibration curve) standards and quality control (QC) samples. Calibration curve standards consisted of a set of non-zero concentrations ranging between 1.013 to 476.760pg/mL were prepared.

 

The QC samples consisted of Drotaverine concentrations of the lower limit of quantification quality control (LLOQQC) 1.013ng/mL, low-quality control (LQC) 2.85ng/mL, middle-quality control (MQC) 276.521 ng/mL and high-quality control (HQC) 376.6404ng/mL were prepared. After bulk spiking, 400μL of spiked plasma samples were pipetted out in pre-labeled polypropylene tubes. The calibration curve standards and QC samples were logged in ultra-low temperature deep freezer (temp range: −55°C to − 75°C) except 30 samples each of LQC and HQC, which were transferred for storage in cell frost deep freezer (temp range: −17°C to − 27°C) for the generation of long-term stability at −22°C±5°C. These samples were used for performing the method validation.

 

Preparation of mobile phase and liquid-liquid extraction method:

A buffer solution was prepared by dissolving approximately 630.60mg of ammonium formate in 1000 mL of milli-Q water and 1mL of formic acid was added in the buffer solution. Mobile phase was prepared as the mixture of acetonitrile: Buffer solution in the ratio 50:50, v/v. For bio-analysis, a set of calibration curve standards and/or QC samples were withdrawn from the deep freezer and allowed to thaw at room temperature. 50μL of Drotaverine as an internal standard (approximately 2000.000pg/mL) was added into ria vials and 300μL of plasma was aliquoted from the pre labeled polypropylene tubes into ria vials followed by vortexing the samples. LLE was performed using ethyl acetate as extraction solvent. Briefly, 2.0mL of extraction solvent was added and vortexed for 10 min. Samples were centrifuged at 4000rpm for 5 min at 4°C and flash freezed for approx. 0.2-2 min. The supernatant was decanted off and evaporated to dryness at 40°C (at constant pressure) in nitrogen evaporator. Residue was reconstituted in 500μL of mobile phase and analyzed.

 

Methodology for validation:

Method validation for Drotaverine in human plasma was done following the USFDA guidelines^[14]

 

RESULTS AND DISCUSSION:

Method development:

The objective of the present work was to develop and validate a selective and sensitive method for Drotaverine by LC MS. Furthermore, the sensitivity of the method should be such that it can monitor at least five half-lives of Drotaverine concentration with good accuracy and precision for the analysis of subject samples. Though there are reports on the simultaneous determination of Drotaverine in human plasma and urine^[15,16], the best chromatographic conditions in terms of resolution, analyte response, peak shape and adequate retention were obtained using acetonitrile- and buffer in 50:50 (v/v) ratio as the mobile phase at a flow rate of 1.000 mL/min. The baseline was resolved within 4.5 min. The retention time of Drotaverine was 3.63 min. Use of labeled IS helped in offsetting any possible ion suppression caused by the plasma matrix and also by compensating any inconsistency during extraction.

 

The mass spectra of Drotaverine and IS were recorded in the positive ionization mode as both the compounds are basic in nature due to the presence of perhydro pyridine ring. Using 10.0ng/mL tuning solution, Drotaverine and IS gave predominant singly charged protonated precursor [M + H] + ions at m/z of 286.1 and 290.0 for Drotaverine and IS, respectively in Q1 full scan spectra. Further, frag- mentation of the precursor ion was initiated by providing sufficient nitrogen for collisional activation dissociation and by applying 25.0 psi curtain gas to obtain highly consistent and abundant product ions of Drotaverine and IS at m/z 166.0 as shown in Fig. S1. Other stable product ions were also found at m/z 194, 215 and 229. However, due to superior signal to noise (S/N) ratio the product ion at m/z 166.0 was selected for quantitation. Additionally, to verify the identity of the analyte and IS qualifier transitions were also monitored at m/z 286.1/194.0 for Drotaverine and m/z 290.0/194.1 for IS. Furthermore, to reach an ideal Taylor cone for better spectral response, nebulizer gas pressure was set at 50 psi to get a consistent and stable response. A dwell time of 300 ms was sufficient to generate at least 24 data points for quantitative analysis of Drotaverine and IS. Also, there was no cross talk between the MRMs of Drotaverine and IS which had identical product ions.

 

Besides, the newly developed method presents an efficient, relatively inexpensive and straightforward extraction procedure for precise and quantitative recovery of Drotaverine. On the other hand, the analysis time of 4.5 min was shorter than in methods reported for the determination of Drotaverine.

 

Method validation:

The method has been validated for selectivity, sensitivity, linearity, matrix effect, calibration curve standards and QC samples, precision and accuracy batches. The results of various stabilities i.e. (stock dilution stability at refrigerator temperature and room temperature, standard stock solution stability in refrigerator temperature and room temperature and photo degradation test in light, auto-sampler stability, re-injection reproducibility, freeze-thaw stability, long-term stability at 65°C±10°C and at − 22°C±5°C, reagent stability, bench top stability, dry ice stability, dry extract stability, extended bench top stability, wet extract stability in refrigerator, lipemic and hemolyzed plasma stability), blood stability, effect of potentially interfering drugs (PIDs), dilution integrity, recovery, ion suppression through infusion, ruggedness, robustness and extended batch verification meeting the acceptance criteria as per the US Food and Drug Administration guidelines (Food and Drug Administration, 2001). Selectivity was performed in 8 lots of normal, 4 lots of lipemic and 4 lots of hemolyzed plasma containing K EDTA as an anticoagulant. Sensitivity of the method was determined in six LLOQ samples. For matrix effect, 12 blank samples were processed from 6 normal plasma lots (two aliquots prepared from each plasma lot) and 6 blank samples were processed from 3 lipemic plasma lots and six blank samples were processed from 3 hemolyzed plasma lots respectively. After drying these processed blank samples from each plasma lot were reconstituted with aqueous LQC and aqueous HQC dilution respectively. For comparison of matrix effect same prepared aqueous LQC and HQC dilution were used and six replicates were injected from each prepared aqueous LQC and HQC. Matrix effect was calculated as per the following formula:

 

% Matrix Effect = (1-mean of matrix effect) X 100

 

The precision of the assay was calculated as percent coefficient of variation (CV) over the concentration range of LLOQQC, LQC, MQC and HQC samples respectively. The accuracy of the assay was calculated as the ratio of the calculated mean values of the LLOQQC, LQC, MQC and HQC samples to their respective nominal values. The data of three precision and accuracy batches were subjected for goodness of fit analysis. The back-calculated concentrations of calibration curve standards using 1/x and 1/x weighing were considered for finding the best fit for regression. Linearity was calculated using a regression equation with a weighting factor of 1/x for the drug to IS concentration to produce the best fit for the concentration detector response relationship for Drotaverine. Stock solution and stock dilution stability in the refrigerator for Drotaverine and internal standard was carried out for 11 days while stock solution and stock dilution stability at room temperature was carried out for 72 h. Photo degradation test of analyte and IS was performed for 72 h in light. For all the aqueous related stability studies, two aqueous mixtures were prepared, one from the stability standard stock solution and the other from fresh standard stock solution (comparison stock). Six replicates of aqueous mixture from each, stability stock and comparison stock were injected. The response of stability sample was corrected using a correction factor.

 

Correction Factor = (Conc. Of fresh Standard Sol.)/ (Conc. Of Stability Standard Sol.)

 

Aqueous recovery comparison samples (LQC, MQC, and HQC) were prepared by adding 6μL each of aqueous dilution of Drotaverine from respective QC samples, 50 μL of internal standard dilution (~2000.000pg/mL) and 444μL of mobile phase (representing 100% extraction). The aqueous samples (LQC, MQC, and HQC) of Drotaverine were compared against 6 sets of processed LQC, MQC, and HQC samples. Recovery of internal standard was compared at LQC, MQC, and HQC level.

 

% Recovery= (Mean peak area response of extracted sample)/(Corrected mean peak area response of unextracted) X100

 

The effect of PIDs i.e. ibuprofen, caffeine, acetaminophen and acetyl salicylic acid on Drotaverine analysis was performed by spiking PID's at their approximately C concentration in the LLOQ sample in triplicate.

 

Bench top stability was determined for 12 h using six sets each of LQC and HQC samples while extended bench top stability was determined in spiked samples to assess the stability of Drotaverine at each step of extraction. The freeze-thaw stability was determined for five freeze-thaw cycles. Six sets of LQC and HOC samples were analyzed after five freeze-thaw cycles. Long-term stability (at −65°C±10°C and −22°C±5°C) was carried out in plasma for 32 days by using six sets of LQC and HQC. Dry extract stability was carried out by processing six sets of LQC and HQC, stored at −22°C± 5°C without reconstitution while wet extract stability was carried out by processing the six sets of LQC and HQC, stored at 2-8°C after reconstitution. The samples of wet extract and dry extract stabilities were analyzed after 75 h storage. All stability QC's were analyzed against the freshly spiked calibration curve standards and six sets of freshly spiked LQC and HQC (prepared from the freshly weighed stock solution) to calculate the % change between the stability QC's and Comparison QC's.

For robustness six sets of LQC and HQC were analyzed against a calibration curve standards at different chromatographic conditions, i.e., robustness experiment was performed at different column temperatures (38°C and 42°C), at different flow rates (0.950mL/min and 1.050mL/min) and at different mobile phase compositions acetonitrile: (10 mm ammonium formate buffer: Formic acid: 99.9:00.1 v/v) 48:52 v/v and 52:48 v/v. To evaluate ruggedness, precision and accuracy batch was processed against calibration curve standards and analyzed by a different analyst using the different column and different sets of solutions.

 

The scanning and acquisition of the parent and the product ions for drotaverine was performed by continuous infusion (5μL/min) of drotaverine and internal standard one by one through a Harvard syringe pump and sorting out the appropriate polarity of ions [M − H] in positive ion mode. The parent ion and product ion mass spectra of the [M − H] ions of are shown in Figure 2.

 

Figure. 2: The parent and product ion mass spectra of the [M − H] ions of Drotaverine

 

Results obtained from the several combinations showed that acetonitrile: (10mM ammonium formate buffer: formic acid: 99.9:00.1 v/v) 50:50 v/v serves the desired purpose with utmost effectiveness. Than source temperature has been increased to 600°C for getting the better spray within the ionization source.

 

Selectivity and matrix effect:

Figure 3 shows typical MRM chromatograms of a blank plasma sample, a plasma sample spiked with drotaverine at the LLOQ (20.013pg/mL) and a plasma sample from a healthy volunteer 2.0 h after the oral administration of the drotaverine tablet. No significant interference was observed from endogenous substances at the RTs of the analyte and internal standard in normal, hemolyzed and lipemic plasma.

 

Figure.3: Representative multiple reaction monitoring chromatograms of (a) Blank plasma sample, (b) A plasma sample spiked with Drotaverine at the lower limit of quantification (c) A plasma sample from a healthy volunteer at 2.0 h (left panel of the figure showing the drotaverine and right panel of the figure showing the internal standard.

 

Linearity:

The LOD is defined as the lowest concentration of an analyte that the bioanalytical procedure can reliably differentiate from background noise while LLOQ is defined as the lowest amount of an analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The lowest standard on the CC is accepted as the LOQ if the analyte response at the LLOQ is at least 5 times the response compared with the processed blank response and the analyte peak (response) should be identifiable, discrete and reproducible with a precision of 20% and accuracy of 80-120%. The correlation coefficients (r) were greater than 0.99 over the concentration range of 0.993ng/mL to 498.245ng/mL.

 

Table.1: Back calculated concentrations of CC standards for drotaverine

			Back Calculated Concentration (ng/mL)		Calibration Standards		Accuracy and Precision
			Batch ID
STD ID	Nominal Conc.	Acceptance Range	P and A-I	P and A-II	Mean	±S.D.	%C.V.	% Accuracy
STD-1	1.013	1.216	0.993	0.980	0.987	0.00919	0.9	97.4
		0.810
STD-2	2.026	2.330	2.016	2.146	2.081	0.09192	4.4	102.7
		1.722
STD-3	4.768	5.483	4.584	4.853	4.719	0.19021	4.0	99.0
		4.052
STD-4	23.838	27.414	22.620	21.461	22.041	0.81954	3.7	92.5
		20.262
STD-5	47.676	54.827	44.423	46.836	45.630	1.70625	3.7	95.7
		40.525
STD-6	95.352	109.655	105.174	109.013	107.094	2.71458	2.5	112.3
		81.049
STD-7	190.704	219.310	208.648	204.643	206.646	2.83196	1.4	108.4
		162.098
STD-8	286.056	328.964	289.612	307.257	298.435	12.47690	4.2	104.3
		243.148
STD-9	381.408	438.619	398.900	391.606	395.253	5.15764	1.3	103.6
		324.197
STD-10	476.760	548.274	498.245	477.240	487.743	14.85278	3.0	102.3
		405.246
r			0.9988	0.9981

 

Accuracy and Precession:

The precision of the analytical method describes the closeness of repeated individual measures of analyte and is defined as the ratio of standard deviation/mean (%). Precision is expressed as the CV. Precision should be demonstrated for the LLOQ, low, medium and high QC samples, within a single run and between different runs, i.e. using the same runs and data as for the demonstration of accuracy. The accuracy of an analytical method describes the closeness of the determined value obtained by the method to the nominal concentration of the analyte (expressed in percentage). Accuracy should be assessed on samples spiked with known amounts of the analyte, the QC samples. Table 1 summarizes back calculated concentrations of CC standards for drotaverine whereas Table 2 represents the intraday precision and accuracy data.

 

Table.2: Intraday validation

QC ID	Actual Conc. (ng/mL)	Mean Values
		P and A-I	P and A-II	MEAN	Standard Deviation	%CV	% Accuracy
HQC	376.640	376.9241	380.7500	378.8371	2.705	0.71	100.583
MQC1	276.521	277.8235	278.3773	278.1004	0.392	0.14	100.571
LQC	2.850	2.7761	2.8118	2.7939	0.025	0.90	98.033
LLOQ QC	1.013	1.0379	1.0470	1.0424	0.006	0.62	102.944

Table.3: Stability data of Drotaverine in processed QC samples for different stability activities at different conditions

Stability Experiment	Nominal Concentration ng/mL	Mean	Precision	Accuracy	Percentage of Stability
Bench top	2.850	2.866	5.3	93.4	98.6
	376.640	389.087	1.3	110.5	102.1
Freeze thaw	2.850	2.64	3.6	93.6	105
	376.640	380.298	3.8	109.9	97.2
Processed sample	2.850	2.789	4.7	94.2	101.4
	376.640	377.95	2.3	109.9	98.1
Auto injector	2.850	2.95	3	93.6	104.1
	376.640	379.46	2.8	109.6	99.1

 

Stability and other parameters:

Table 3 represents stability data and shows there were no stability-related issues that might cause problems in the application of the assay to PK studies.

 

CONCLUSION:

A method was developed for quantification of Drotaverine in plasma by using LC-MS equipped with ionization spray and is operated in positive ion mode using multiple reaction monitoring and fully validated. The advantage of this method is more economic, sensitive and preventing the contamination and interference peaks from various unknown sources which were well separated in liquid-liquid extraction. It is rugged and robust technique to attain incurred sample reanalysis. The current method has shown acceptable precision and adequate sensitivity for the quantification of Drotaverine in plasma samples. The sensitivity of Drotaverine was achieved with an LLOQ of 1.013 ng/mL, which has an intraday CV of 0.91% respectively. Many variables related to the electrospray reproducibility were optimized for both precision and sensitivity to obtain these results. The method can be successfully applied to quantify the concentrations of Drotaverine in pharmacokinetic studies.

 

CONFLICTS OF INTEREST:

The authors declare that there are no conflicts of interest

 

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Received on 21.11.2019          Modified on 02.02.2020

Accepted on 03.04.2020         © RJPT All right reserved