INTRODUCTION:

Bioanalytical method development plays an essential role in preclinical and clinical studies. The major part of bioanalysis is mainly concerned with developing a suitable method to determine the concentration of drugs, their metabolites, and/or endogenous substances in the biological matrices such as blood, plasma, serum, cerebrospinal fluid, urine, and saliva^1,2. The application of the bioanalytical method is mainly focused on the evaluation of bioavailability, bioequivalence, pharmacokinetic studies of investigating drug molecules and other biological entities in various biological matrixes.^3,4,5,6.

Febuxostat is an orally administered non-purine xanthine oxidase selective inhibitor approved for chronic hyperuricemia management in gout patients.⁷ Xanthine oxidase is needed to oxidize hypoxanthine and xanthine successively to uric acid. Febuxostat, therefore, inhibits xanthine oxidase, reducing uric acid production. Because of its firm binding to the Molybdenum Pterin Site, Febuxostat inhibits both oxidized and reduced forms of xanthine oxidase.⁸

Fig. 1: Febuxostat Structure.

Tolbutamide (Used as Internal Standard) is an oral hypoglycemic sulfonylurea medicine for first-generation potassium channel blockers. If the diet alone is not adequate, this drug can be used for treating type 2 diabetes. Tolbutamide stimulates the pancreatic secretion of insulin.

Selective and sensitive bioanalytical methods for the quantitative evaluation of drugs and their metabolites are critical for the successful biopharmaceutics and clinical pharmacology studies⁹. The validation of the biological method comprising all methods that show that the specifically developed method for quantitative analyte measurement is reliable and reproductive for the intended use in a specific biological matrix¹⁰. The bioanalytical method undergoes many modifications during a drug development process. Each modification should be validated to ensure the proper performance of the bioanalytical method. The objective of method validation is to exhibit the reliability of a particular method developed for the quantitative determination of an analyte in a specific biological matrix^11-16. Literature survey reveals that few analytical methods are done for the determination of Febuxostat by HPLC in blood plasma.¹⁷

MATERIALS AND METHODS:

Chemicals and Reagents:

The active pharmaceutical ingredient (API) of Febuxostat (Purity 98.33%) was obtained from AKUMS Drugs and Pharmaceutical Ltd... Moreover, Tolbutamide was obtained from Shubham Biopharma, which is used as an Internal Standard (IS). Acetonitrile (HPLC grade), Formic Acid (HPLC grade), DMSO (HPLC grade) were purchased from Merck Pvt. Ltd, Mumbai, India. Water was purified by a Milli-Q gradient system of Millipore (Elix, Milli-Q A10 Academic, Bedford, MA, USA) until a resistively of 18.2 MΩ.cm was achieved. The blank human plasma with EDTA-K3 anticoagulant was collected from the Clinical Pharmacological Unit (CPU) of TAAB Biostudy Services, Kolkata, India.

Instrumentation:

The liquid chromatographic system consists of Shimadzu series LC-20 AD Binary pump, CTO-10 AS VP Column oven, SIL 20 AC Autosampler, andCBM-20A Lite System Control Compartment. The LC-MS/MS system API 2000 with triple quadrupole tandem mass spectrometer (Applied Biosystems/MDS SCIEX, Instruments) was used for the quantitative determination of Febuxostat and Tolbutamide (IS) in human plasma. Data acquisition, processing, and quantitation were performed with Analyst 1.6.3 software (Applied Biosystems/MD SCIEX).

Chromatographic conditions:

Chromatographic separation was done on an Phenomenex Kinetex C18; 50x3mm, Particle Size-5µm column. The Mobile phase used for the elution of the analyte was Pump A: 0.1% Formic Acid in Water (Milli-Q) and Pump B: 0.1% Formic Acid in Acetonitrile. The flow rate was set at 0.5mL/min. The injection volume was 10µL and the total run time was 7.0 minutes. The column temperature was not applicable in this way, while the autosampler temperature was maintained at 15^oC.

Mass spectrometry

Turbo electrospray ionization (ESI) interface with multiple reaction monitoring (MRM) was used to acquire the mass spectra of the compounds. Ions were measured in negative ionization mode. The tuning parameters were optimized by injecting 100 ng/mL of a standard solution containing Febuxostat and Tolbutamide (IS) at 10µL/min. through an external syringe pump directly connected to the mass spectrometer. The applied high voltage was -4.5 Kv to the spray needle. The instrument was programmed for a scan dwell time of 100.00 msec. The MRM transitions of m/z 315.100 ®271.100, m/z 269.200 ®169.700 were used to measure Febuxostat and Tolbutamide (IS), respectively (Table 1). The source temperature of turbo ion spray was set at 400^oC, and the turbo ion spray voltage was set at -4500.00 V. The value set of nebulizer gas (GS1) was 45.00 p.s.i. Moreover, the turbo ion-spray gas (GS2) value was set at 45 p.s.i. The curtain gas (CUR) and the collision-associated dissociation (CAD) gas flow at instruments were 30.00 and 5.00 (arbitrary scale), respectively (Table 2). Figure 2.A: Q1 Scan and Figure 2.B: Q3 (MS2) Scan of the drug Febuxostat.

Table 1: Mass Spectrometric Conditions:

	FEBUXOSTAT	TOLBUTAMIDE (IS)
Precursor (DA)	315.100	269.200
Product (DA)	271.100	169.700
Dwell (ms)	100.00	100.00
DP (V)	-13.00	-18.00
FP (V)	-394.00	-371.00
CEP	-16.40	-15.25
CE (Ev)	-12.00	-28.00
EP (V)	-6.80	-5.70
CXP (V)	-8.50	-11.00

Table 2: Gas/Source Parameters:

CUR (Psi)	30.00
CAD (Psi)	5.00
IS (V)	- 4500.00
TEM (^oC)	400.00
GS1 (Psi)	45.00
GS2 (Psi)	45.00

Figure 2. A: Q1 Scan of Febuxostat

Figure 2. B: Q3 (MS2) Scan of Febuxostat

Preparations of Standard solutions, quality control samples and calibration curve:

The standard solutions were prepared using DMSO containing 1mg/mL of Febuxostat and IS. From this solution, we prepare a stock solution of 1µg/mL for Febuxostat and 15μg/mL for IS. From the drug stock solution, we prepare seven working solutions for calibration standards, they were 125ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/ml, and 8000ng/mL. The solutions are stored at 2⁰ – 8⁰C.

The four types of quality control samples are LLOQ, LQC, MQC, and HQC were prepared from the seven-point standard calibration solutions of Febuxostat. The LLOQ was 125ng/mL., lowest calibration concentration. The LQC was 375ng/mL., middle concentration of second (250ng/mL) and third (500ng/mL) concentrations. The MQC was 3000ng/mL. middle concentration of fifth (2000ng/mL) and sixth (4000 ng/mL) concentrations. The HQC was 6000ng/mL., middle of concentration of sixth (4000ng/mL) and seventh (8000ng/mL) concentrations.

Standard calibration solutions of Febuxostat was prepared by spiking appropriate amounts of analyte and IS in blank human plasma to yield final concentrations. The quality control (QC) samples were prepared at three concentration levels of analyte. Calibration curves were plotted with a peak area ratio of drug and IS on Y-axis and concentration on X-axis.

Sample preparation:

Protein precipitation technique (PPT) is one of the best and simple techniques for plasma extraction. Only 100μl of blank plasma sample was transferred to a 2ml plastic Eppendorf tube. 50μl of drug and 50μl of IS working solutions were spiked into it and vortex for 1 minute. After adding 300μl cold Acetonitrile and vortexing for 10 minutes. After that centrifugation was done at 12000rpm for 5min by cold centrifuge at 4⁰C Then 300µl of supernatant was taken and transferred to autosampler vials for injections.¹⁸

Bioanalytical Method Validation:

The method validation was executed as per USFDA and EMA guidelines.

Selectivity:

Blank plasma from the each healthy human volunteer was collected for establishing selectivity of the method. Selectivity was determined at the LLOQ level means a lower limit of quantification. The acceptance criteria as per the USFDA guideline for selectivity is the area response at the retention time (RT) of the analytes in blank should not be more than 20% compared to the response in LLOQ.¹⁹

Carryover:

The possibility of interference from a run to that of the immediately injected next run was analyzed by injecting blank samples after HQC. According to the USFDA guideline, the carryover from an injection of high concentrations should be < 20% of the area of LLOQ.¹⁹

Sensitivity:

The spiked plasma samples at LLOQ level were injected five times for confirmation of the sensitivity of the method. As per the guideline of USFDA, the area of the analyte peak at LLOQ should be higher than five times the blank's peak area. As per the guideline of USFDA, the accuracy of the injected LLOQ samples should be within ±20%.¹⁹

Linearity:

Linearity was evaluated through regression analysis for the seven-point (125, 250, 500, 1000, 2000, 4000, and 8000ng/mL) standard calibration curve. The standard calibration curve was constructed, taking the drug/IS ratio on Y-axis and concentration on X-axis to determine the coefficient of determination (R²).

Accuracy:

Accuracy of the method was evaluated in five replicates at four different concentrations of LLOQ, LQC, MQC, and HQC. The drug to IS peak area ratio was determined, and the concentration was back-calculated from the line equation (y=mx+c). The deviation of the estimated back-calculated value from the theoretical concentration was determined to calculate the accuracy. As per the guideline of USFDA, the acceptance criteria for accuracy is±15% of the theoretical concentration except at LLOQ, where it should be within ±20%.¹⁹

Precision:

The precision of the analytical procedure was assessed after injecting the spiked plasma samples in five replicates at LLOQ, LQC, MQC, and HQC levels. The %CV of the back-calculated concentrations of the repeated injections was calculated. Intraday precision was assessed by determining %CV of the response of the injections injected on the same day. Additionally, inter-day precision was calculated after determining the %CV of the measured values of the samples injected on different days. As per the guideline of USFDA.¹⁹

Extraction Recovery:

The potential of the sample preparation technique to extract Febuxostat, including IS from the biological sample, was determined by comparing the chromatographic response found in the extracted samples at LQC, MQC, and HQC in three replicates to that of the unextracted samples of the same concentration which correspond 100% recovery.^19,20

Matrix Effect:

Comparison of the chromatographic responses of the post-extracted HQC, MQC and LQC samples with the response of the analytes from neat samples at equal concentrations was done to get the effect of plasma constituents over analyte ionization to determine IS. Similar concentration of analyte and IS concerning the recovery experiment was used to determine the matrix effect.

Robustness:

The robustness of the method was established by making deliberate minor variations in the composition of flow rate and mobile phase.^21-25

Limit of Detection (LOD)and Lower Limit of Quantification (LLOQ):

LOD and LLOQ they were determined at signal to noise ratios by injecting series of dilute solutions with known concentrations.^26,27,28

Stability:

The stability of the drug in plasma was estimated by performing different stability testing like short-term stability, freeze and thaw cycle stability, autosampler stability, benchtop stability, and long-term stability. Blank Human plasma was spiked with the analytes at the concentration of three quality control (LQC, MQC, and HQC) in five replicates followed by extraction and analyzed after completing the suitable storage condition for individual stability test. The stability of the analytes in stock solution was assessed by freshly preparing the QC samples from the stock solution before the study. Short-term stability study was spiked plasma samples were stored at -20°C for 24 hours, followed by extraction and analysis. Freeze and thaw stability study was done by storing samples at -20°C and exposed to three repeated freeze and thaw cycles by freezing at -20°C and thawing at normal laboratory conditions. Autosampler stability study was done by processed samples which were analyzed by injecting the samples after keeping 24 hours inside the autosampler maintained at 15°C. Benchtop stability study was performed by analyzing the plasma samples spiked with the analytes after keeping 24 hours on bench top at room temperature. Long-term stability study was spiked plasma samples were stored at -20°C for 45 days, followed by extraction and analysis. All the stability samples were compared against freshly spiked QC samples at LQC, MQC, and HQC level. As per the guideline of USFDA the stability studies, the accuracy at each level should be within ±15%.¹⁸

RESULTS AND DISCUSSIONS:

Selectivity:

The chromatograms showed no response in any of the blank samples, indicating an absence of any interference of plasma components at the similar RT of Febuxostat. For this reason, the developed method met the selectivity requirement as the RT in blank samples' peak area was ≤20% compared to the area in LLOQ for the analyte. Figure 3.A is the representative chromatograms of blank with IS. Figure 3.B represents a chromatogram of LLOQ with IS.

Table 3: Inter-day-accuracy:

Quality control	Concentration (ng/mL)	Run	Mean found (ng/mL)	SD	CV (%)	Accuracy (%)
LLOQ	125	1	115.59	4.70	4.07	92.47
		2	136.51	10.66	7.81	109.20
		3	116.92	3.87	3.31	93.54
LQC	375	1	375.51	11.10	2.96	100.14
		2	411.43	14.10	3.43	109.72
		3	395.69	6.43	1.63	105.50
MQC	3000	1	2905.57	45.49	1.57	96.85
		2	3097.87	89.77	2.90	103.26
		3	3058.09	54.72	1.79	101.94
HQC	6000	1	5372.53	92.55	1.72	89.54
		2	5724.87	130.04	2.27	95.41
		3	5603.54	120.21	2.15	93.39

Table 4: Intraday-accuracy:

Quality control	Concentration (ng/mL)	Mean found (ng/mL)	SD	CV (%)	Accuracy (%)
LLOQ	125	121.08	5.66	4.67	96.86
LQC	375	390.78	5.20	1.33	104.21
MQC	3000	3129.48	120.17	3.84	104.32
HQC	6000	5549.49	84.08	1.52	92.49

Table 7: Stability Studies:

Stability	Quality control	Concentration (ng/mL)	Mean found (ng/mL)	Accuracy (%)
Freshly Thawed	LQC	375	379.18
	MQC	3000	3095.64
	HQC	6000	5672.33
Bench top (24 Hours)	LQC	375	400.90	105.73
	MQC	3000	3109.27	100.44
	HQC	6000	5709.30	100.65
Autosampler (24 Hours)	LQC	375	394.69	104.09
	MQC	3000	3043.63	98.32
	HQC	6000	5956.82	105.02
Freeze-thaw (3 Cycles)	LQC	375	409.24	107.93
	MQC	3000	3038.02	98.14
	HQC	6000	5416.12	95.48
Short term (24 Hours)	LQC	375	386.29	101.88
	MQC	3000	2980.53	96.28
	HQC	6000	5493.94	96.86
Long term (45 Days)	LQC	375	383.06	101.02
	MQC	3000	3218.80	103.98
	HQC	6000	5956.80	105.02

CONCLUSION:

In this present study, a simple bioanalytical method was developed for the detection and quantification of Febuxostat in human plasma by using LC-ESI-MS/MS. The sample preparation procedure consists of a simple protein precipitation technique (PPT) using cold Acetonitrile. The developed method is very cost-effective and also was found to have accepted in terms of various parameters like selectivity, accuracy, precision, and stability performed as per the guideline that is USFDA bioanalytical method validation. Therefore, this study definitely will have a significant contribution to the field of bioanalytical research and will be superior enough to apply in comparative pharmacokinetic studies in the future. The entire study to analyses plasma drug concentration was designed with proper validation to find the vital pharmacokinetic parameters of the drug Febuxostat. The method can be suitably used by the researchers to calculate pharmacokinetic parameters and as well as bioavailability of the drugs in human volunteers. And the routine analysis of Febuxostat in pharmaceutical dosage forms for routine application in quality control laboratories.

ACKNOWLEDGEMENT:

The authors want to acknowledge M/S, TAAB Biostudy Services, Kolkata-700032, India, for providing blank human plasma samples and the necessary instrumental facilities to complete the work.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 23.07.2020 Modified on 19.08.2020

Research J. Pharm. and Tech. 2021; 14(8):4060-4066.

DOI: 10.52711/0974-360X.2021.00703

	Diluent Sample			In Plasma Sample
Quality Control	LQC	MQC	HQC	LQC	MQC	HQC
Concentration (ng/mL)	375	3000	6000	375	3000	6000
Mean found (ng/mL)	411.16	3104.23	5472.40	402.68	3029.61	5452.22
% Recovery				97.94	97.60	99.63

		Extracted Blank Plasma Sample			Aqueous Sample			% OF ME
Quality Control	Concentration (ng/mL)	Mean found (ng/mL)	SD	CV (%)	Mean found (ng/mL)	SD	CV (%)	% OF ME
LQC	375	374.56	18.73	5.04	404.40	4.17	1.03	92.60
MQC	3000	3017.20	25.43	0.84	3202.72	20.52	0.64	94.21
HQC	6000	5481.16	130.42	2.38	5996.19	45.01	0.75	91.42