INTRODUCTION:

Chemically MAT (Fig 1) is, 5-[(3,5-dimethylphenoxy)methyl]-1,3-oxazolidin-2-one [1]. It is a skeleton muscle relaxantused in the short-term treatment of painful muscle spasms [2]. It isgenerally used alone or in combination with diclofenac. Several analytical methods have been reported for the estimation of MAT, includes estimation of MAT in pharmaceutical formulation by UV spectroscopy alone [3] and in combination with diclofenac [4,5,6], quantitation of MAT in different physiological fluids by UV spectroscopy [7], estimation of MAT in pharmaceutical formulations by HPLC [8,9,10,11] and estimation of MAT in combination with diclofenac by HPTLC [12].

Along with this, several analytical methods have been reported for the estimation of MAT in human plasma by LC-MS [13,14,15,16,17] and by high-throughput LCMS/MS [18] as well estimation of MAT in rat plasma (19) by LCMS and by LCPDA [20].

Fig1: Chemical structure of MAT

In the present paper, a simple and rapid HPLC-UV method has been describedfor the estimation of MAT in spiked human plasma. MAT was extracted from human plasma using liquid-liquid extraction with suitable organic solvent. Further, the method was validated as per US-FDA Bioanalytical Method Validation: Guidance for Industry May 2018 [21].

MATERIALS AND METHODS:

Chemicals and Reagents:

Pharmaceutical grade MAT was supplied as a gift sample from Amnel pharmaceuticals Pvt Ltd., Ahmedabad, India. Blank human plasma sampleswere procured as a gift sample from Dr. Vasantrao Pawar Medical College Hospital and Research Centre, Nashik, India. The pooled plasma sample was prepared by mixing thoroughly obtained plasma samples from six different sources. Methanol. acetonitrile used in the analysis were of HPLC grade and other chemicals were of analytical grade. All chemicals were purchased from Merck Ltd., Mumbai, India. Double distilled water used in the analysis was prepared freshly and used after filtration through Durapore 0.45m × 47 mm membrane filter papers, purchased from Milipore India Pvt Ltd., Bengaluru, India.

Instrumentation and chromatographic condition:

HPLC analyses were carried out using a JASCO HPLC system consists of dual PU-2080 plus pumps, UV-2075 UV multi-channel UV detector and equipped Rheodyne (7725i) injection system with 100mL sample loop. Data obtained from the system were collected and processed using Borwin Chromatography software (version 1.50).

All chromatographic separations were carried out on Phenomenex Hyperclone C18 (BDS) column (250 × 4.6 mm, 5m) using a mixture of acetonitrile: water (50:50%, v/v) as a mobile phase in an isocratic mode at a constant flow rate of 1mL/min. All eluents were detected at the 217nm, the absorption maxima of the drug.

Liquid-liquid extraction:

An aliquot of 1mL of pooled human plasma was taken in a stoppered glass tube of capacity 20mL. To this tube, 100mL of 100mg/mL of MAT, 100mL of 100mg/mL of IS and 200mL of 1% formic acid was added. The resulting solution was vortex mixed for 3min. Further, the extracting organic solvent, TBME was addedinto it and again vortex mixed for 5 min. The tubes were centrifuged at 4ºC, for 10min at 3000 rpm. The organic layer was transferred to another tube and the solvent was evaporated under the stream of nitrogen. The residue was further reconstituted with 500mL of the mobile phase and the 100mL was injected into the HPLC system.

Preparation of calibration curve (CC) standards and Quality Control (QC) samples:

The standard stock solution of MAT was appropriately diluted with methanol to get six different working standard solutions having concentrations of 1, 2, 3, 4, 8, 10, 16, 20, 32 and 40mg/mL. To obtain the calibration curve (CC) standard, 1mL aliquots of the pooled blank plasma sample were taken in nine stoppered glass tubes of 20 mL capacity, in each tube, 100mL of working standard solution of MAT, 100mL of 100mg/mL of IS and 200mL of formic acid was added to get results CC standards of 0.1, 0.2, 0.3, 0.4, 0.8, 1.0, 1.6, 3.2 and 4.0 mg/mL. All CC standards were similarly processed as per the procedure described in liquid-liquid extraction and finally injected into the HPLC system under optimized chromatographic conditions in six replicates. The chromatograms of CC standards were processed to obtain the peak area of MAT and IS and the retention factor was calculated for each CC standard by calculating the ratio of area of MAT to the area of IS.

Along with the CC standards, QC samples consisting of 0.3mg/mL of LQC, 1.6mg/mL of MQC and 3.2mg/mL of HQC were prepared.

Selection of calibration model:

Data obtained from calibration experiments were subjected to unweighted and weighted linear regression with the weighting factor of 1/x, 1/Öx and 1/x². From the tested regression models, the appropriate model was selected with the minimal % RE and heteroscedaticity in the linear range and was used for further calculations.

Validation:

The developed method was validated as per the US-FDA guidelines Bioanalytical Method Validation: Guidance for Industry, May 2018 with respect to selectivity, accuracy, precision, recovery, stability and carry-over.

Selectivity was carried out at lower limit of quantitation (LLOQ) at the concentration of 0.1mg/mL against blank response of plasma obtained from six different sources. Accuracy and precision were carried out by analysing and processing five replicates of LQC, MQC and HQC over five days. The accuracy was estimated as the mean % RE and precision was measured in terms of % RSD. Recovery was calculated by comparing peak areas of processed QC samples to the standard dilutions. Stability studies conducted includes, stability at room temperature, stability at -20°C, bench-top stability and freeze-thaw stability. The % nominal and % RSD were calculated. Further, the carry-over experiment was conducted by injecting a series of sample injections in order of blank solution, unextracted upper limit of quantitation (ULOQ), blank solution, extracted blank matrix, extracted ULOQ and extracted blank matrix.

RESULTS AND DISCUSSION:

Different compositions of mobile phases were tried to separate the MAT and IS from the plasma components. It was found that acetonitrile: water (50:50 %, v/v) gave adequate retention and resolution in an isocratic mode at a flow rate of 1mL/min at 217nm. The retention time for MAT and IS was found at 5.4min and 6.9min, respectively. Different organic solvents (ethyl acetate, chloroform, TBME and diethyl ether) were used to extract the MAT and IS from plasma samples. It was found that MAT and IS were well extracted with TBME compared to other organic solvents. Also, the recovery of MAT and IS was found to improved when plasma samples was acidified with 1 % formic acid along with TBME. Hence, it was decided to add 200mL of 1 % formic acid in each sample. The results of % recovery of MAT in different organic solvents are depicted in Table 1 and the chromatogram of blank plasma extracted with TBME and the representative chromatogram of MAT and IS extracted in TBME are shown in Fig 2 and Fig 3, respectively. Under mentioned liquid-liquid extraction, the recovery of MAT and IS were found to be 50.68 % and 43.99%, respectively. For the highest concentration of MAT (4mg/mL), when different concentrations of IS were analysed, it was found that 10mg/mL of IS gave the peak area between 30-60% to that of the highest CC standard peak area of MAT.

Table 1: Recovery of MAT and IS in different organic solvents

Organic solvent	% Recovery of MAT	% Recovery of IS
n-hexane	5.06	8.26
Chloroform	30.67	11.25
Dichloromethane	31.87	14.75
Ethyl acetate	34.84	15.26
Diethyl ether	37.64	17.56
Ter-butyl methyl ether (TBME)	40.63	20.56
TBME with 1 % formic acid	50.68	43.99

The data from the calibration experiment revealed the increase in the standard deviation (±SD) with increasing concentrations (Table 2) suggests the need of weighted linear regression. When the obtained calibration data were subjected to weighted linear regression with weighting factor of 1/x, 1/Öx and 1/x², it was observed that, the weighted linear regression with 1/x weighting factor shows minimal % RE and F values. Hence, it was decided to use a weighted linear regression with 1/x weighting factor for further calculations with the regression equation of y = 0.00014 + 0.01459x.

Table 2: Calibration Curve (CC) standard data of MAT

CC No.	Concentration in mg/mL	RF (mean ± SD) (n=6)
1	0.1	0.0278±0.0011
2	0.2	0.0404±0.0012
3	0.3	0.0615±0.0016
4	0.4	0.0757±0.0028
5	0.8	0.1131±0.0029
6	1.0	0.1578±0.0039
7	1.6	0.2425±0.0047
8	2.0	0.2895±0.0051
9	3.2	0.4760±0.0167
10	4.0	0.5645±0.01967

The method was selective at LLOQ concentration of 0.1 µg/mL, as the responses of the plasma samples from six different sources were found less than 20 % of the response obtained for LLOQ (Table 3).

Table 3: Blank responses and LLOQ peak areas of MAT

Sr. No.	Blank response (µV.sec)	Peak areas at LLOQ (µV.sec)	% peak area in blank
1	1140	17394	6.50
2	1053	13803	5.91
3	1004	26104	3.84
4	1215	27560	4.40
5	1025	21633	4.73
6	1016	19979	5.08

Also, the chromatogram of blank plasma extract does not show any significant peak at the retention times of MAT and IS. The results of accuracy and precision studies are shown in Table 4, the method was found accurate and precise as the obtained RE and %RSD were found within ± 15 and less than 15%.

Table 4: Results of accuracy and precision studies

QC Level	Conc. Added (mg/mL)	Intra-day			Inter-day
QC Level	Conc. Added (mg/mL)	Mean conc. (n=1)	% RE	% RSD	Mean conc. (n=1)	% RE	% RSD
LQC	0.3	312.76	-3.05	6.5	284.99	-2.33	4.14
MQC	1.6	1596.41	-0.22	2.0	1570.68	-1.83	2.35
HQC	3.2	3141.35	-14.38	8.2	3002.72	-5.89	2.45

Table 5: Stability study o MAT at room temperature and at -20°C

QC Levels	Stability at Room Temperature						Stability at -20°C
	% Nominal			% RSD			% Nominal			% RSD
	2 h	4 h	6 h	2 h	4 h	6 h	10days	20days	30days	10days	20days	30days
LQC	99.96	95.83	100.50	1.17	6.1	2.8	99.41	95.74	99.00	1.40	8.18	3.23
HQC	100.50	101.30	100.10	1.22	1.70	4.06	99.06	99.72	100.30	2.64	1.39	1.17

Table 6: Bench-top and freeze thaw stability studies of MAT

QC Level	Bench-top stability		Freeze thaw stability
	% Nominal	% RSD	% Nominal			% RSD
	% Nominal	% RSD	FT 1	FT 2	FT 3	FT 1	FT 2	FT 3
LQC	105.44	4.4	99.88	97.74	99.63	1.12	3.21	0.36
HQC	99.96	1.93	100.05	99.65	99.75	0.46	2.93	3.47

The results of stability studies concluded that the MAT remained stable in human plasma samples under the studied stability cycles. The results of stability studies are depicted in Table 5 and Table 6.

When carry-over study was conducted under mentioned sequence for MAT, it was observed that the response of mean extracted ULOQ was £ 20 % and £ 5 % of response of extracted IS. The results of carry-over test are presented in Table 7.

Table 7: Results for carry-over test

Sr. No.	Sample	Peak Areas (µV.sec)
1	Blank DI	0	0
2	AQ-ULOQ	669220.750	636385.00
3	BLANK DI	0	0
4	AQ-ULOQ	644226.500	615632.500
5	Blank DI	0	0
6	Blank plasma	0	0
7	Extracted ULOQ	300142.598	269848.355
8	Blank plasma	0	0
9	Extracted ULOQ	330272.250	342395
10	Blank plasma	0	0

ACKNOWLEDGEMENT:

Authors are thankful to the management and trustees of the Bhujbal Knowledge City, MET’s Institute of Pharmacy, Nashik for proving necessary analytical facilities, Dr. Vasantrao Pawar Medical College Hospital and Research Centre, Nashik for proving gift sample of blank human plasma and to Amnel Pharmaceuticals Pvt Ltd, Ahmedabad for proving gift sample of MAT.

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Received on 01.08.2019 Accepted on 20.09.2019

Asian J. Pharm. Ana. 2019; 9(4):210-214.

DOI: 10.5958/2231-5675.2019.00035.8