Rapid RP-HPLC Method for Simultaneous estimation of Umeclidinium and Vilanterol in human plasma

 

G. Raveendra Babu^1,2*, M. Sowjanya³, M. Ramayyappa², Md. Abdul Karishma²,

V. Bharathi², G. Renuka Swathi², Ch. Renuka², G. Gopala Krishna², M. Lavanya²

¹Department of Pharmaceutical Analysis, QIS College of Pharmacy, Ongole - 523001, A.P., India.

²Department of Pharmaceutical Analysis, A.K.R.G. College of Pharmacy, Nallajerla - 534112, A.P., India.

³Department of Chemistry, Vijaya Teja Degree College, Addanki - 523201, A.P., India.

 

ABSTRACT:

A simple, precise, accurate method was developed for the estimation of Umeclidinium andVilanterol in human plasma using Empagliflozin as internal standard by RP-HPLC (Reverse phase-High performance Liquid Chromatographic) technique. Chromatographic conditions used are stationary phase KromasilC18 (250 x 4.6 mm, 5m)coulmn, mobile phase of 0.01N di sodium hydrogen phosphate (pH: 3.0): Acetonitrile in the ratio of 65:35(v/v) and flow rate was maintained at 1.0ml/min, detection wave length was 215nm, column temperature was set at 30^oC and diluent was mobile phase. Retention time of Umeclidinium and Vilanterol and were found to be 2.471min and 3.303min. %CV of the Umeclidinium and Vilanterol were found to be 0.33% and 0.43%. %Recovery was obtained as 99.34% and 99.029%. The linearity concentration is in the range of 1.25-50µg/mL of Umeclidinium, 0.25-10.0µg/mL of Vilanterol respectively. The lower limits of quantification were 1.25µg/mL of Umeclidiniumand  0.25µg/mL of Vilanterol which reach the level of both drugs possibly found in human plasma. Further, the reported method was validated as per the ICH guidelines and found to be well within the acceptable range.

 

 

 

1. INTRODUCTION: 

Umeclidinium is chemically diphenyl-[1-(2-phenylmethoxyethyl)-1-azoniabicyclo[2.2.2]octan-4-yl]methanol. It is in a class of medications called anticholinergics. Umeclidinium is a long-acting muscarinic antagonist (LAMA) used as maintenance treatment for symptoms of chronic obstructive pulmonary disease (COPD). It is available as a once-daily inhalation monotherapy or as a fixed-dose combination product with the long-acting beta2-agonist vilanterol. Its use has been shown to provide clinically significant, sustained improvements in lung function^1-3.

 

Vilanterol is 4-[(1R)-2-[6-[2-[(2,6-dichlorophenyl)methoxy]ethoxy]hexylamino]-1-hydroxyethyl]-2-(hydroxymethyl)phenol. It isa long-acting beta-2 adrenergic agonist, with bronchodilator activity. Upon administration, vilanterol stimulates beta-2 adrenergic receptors in the lungs, thereby activating the enzyme adenylate cyclase that catalyzes the conversion of adenosine triphosphate (ATP) to cyclic-3',5'-adenosine monophosphate (cAMP)^4-6.

 

The combination of umeclidinium and vilanterol is used to control wheezing, shortness of breath, coughing, and chest tightness caused by chronic obstructive pulmonary disease (COPD; a group of diseases that affect the lungs and airways, which includes chronic bronchitis and emphysema). To the best of our knowledge and comprehensive survey, Umeclidinium, and vilanterol are analyzed individually using UV, Fluorometric methods and HPLC^7-19 methods individually but in combination with other drugs, but were never determined before by chromatographic techniques in biological samples despite their wide usage. As such, the present work introduces a simple, very rapid, reproducible, and sensitive chromatographic method for the determination of these drugs in human plasma²⁰.

 

Fig. 1: Chemical structure of Umeclidinium

 

Fig. 2: Chemical structure of vilanterol

 

Fig. 3: Chemical structure of Empagliflozin.

 

2. EXPERIMENTAL:

2.1. Apparatus:

Waters HPLC 2695 system with a Kromasil C18 (250 x 4.6mm, 5m) column, a diodearray- detector (DAD) absorbance detector, equipped with quaternary pumps, and connected to a PC computer loaded with empower software was used.LABINDIA UV-Vis Double Beam spectrophotometer with matched 1-cm quartz cells and connected to windows-compatible computer using UV Win 5 Software v6 was used for spectral studies.

 

2.2. Materials and Reagents:

All solvents and reagents were of HPLC-analytical grade (methanol, acetonitrile, potassium dihydrogen phosphate, and orthophosphoric acid were provided by Fisher Scientific).Umeclidinium and VilanterolAPI  was obtained as a gift sample from Spectrum pharma research solutions. Standard solutions of 200μg/mL were prepared by dissolving 0.01mg of each pure drug in 50mL of the mobile phase.Mobile phase is selected to be 0.01N di sodium hydrogen phosphate (pH: 3.0): acetonitrile in the ratio of 65:35(v/v) and a flow rate was maintained at 1.0ml/min, detection wave length was fixed at 215nm, column temperature was set to be 30^oC. The mobile phase was filtered and degassed using a 0.45-μm membrane filter.The human plasma was kindly provided by Deccan Pathological labs, Hyderabad and was tested to be drug and disease-free. The plasma was kept frozen before use and was then stored either at −4 °C between uses or at −20°C for freeze–thaw cycle stability studies.

 

2.3. Procedures:

Preparation of Umeclidinium Stock solution (2500 µg/ml):

Take 125mg of Umeclidinium in 50ml volumetric flask and make the volume with diluent to produce 2500 µg/ml.

 

Preparation of Umeclidinium Spiking Solutions:

From the above Umeclidinium stock solution 0.05ml, 0.1ml, 0.15ml, 0.6ml, 1.0ml, 1.2ml, 1.6ml and 2.0ml was pipette and transferred to 8 individual 10ml volumetric flask and make up the volume up to the mark with diluent to produce 12.5µg/ml, 25µg/ml, 37.5µg/ml, 100µg/ml, 250µg/ml, 300µg/ml, 400µg/ml and 500µg/ml.

 

Calibration standards and quality control (QC) samples were prepared by spiking blank plasma with working stock dilutions of analytes to produce 1.25µg/ml, 2.5 µg/ml, 3.75µg/ml, 10.0µg/ml,  25ng/ml, 30.0µg/ml, 40.0 µg/ml and 50.0µg/ml.

 

Preparation of Vilanterol Stock solution (500 µg/ml):

Take 25mg of Vilanterol in 50ml volumetric flask and make the volume with diluent to produce 500µg/ml.

 

Preparation of Vilanterol Spiking Solutions:

From the above Vilanterol stock solution 0.05ml, 0.1ml, 0.15ml, 0.6ml, 1.0ml, 1.2ml, 1.6ml and 2.0ml was pipette and transferred to 8 individual 10ml volumetric flask and make up the volume up to the mark with diluent to produce 2.5µg/ml, 5.0µg/ml, 7.5µg/ml, 20.0 µg/ml, 50.0µg/ml, 60.0µg/ml, 80.0µg/ml and 100.0 µg/ml.

 

Calibration standards and quality control (QC) samples were prepared by spiking blank plasma with working stock dilutions of analytes to produce 0.25µg/ml, 0.5µg/ml, 0.75µg/ml, 2.0µg/ml, 5.0µg/ml, 6.0µg/ml, 8.0 µg/ml and 10µg/ml.

 

Preparation of internal standard Solution (Empagliflozin):

Stock-1: Take 50mg of Empagliflozin in 100ml volumetric flask and make up the volume with diluent to produce 500µg/ml.

 

Stock-2: From the above solution, take 1ml of solution into 10ml volumetric flask and make up the volume with diluent to produce 50µg/ml solutions.

 

Final concentration:

From the above solution, take 0.5ml of solution and spiking blank plasma with working stock dilutions of analyte to produce 10µg/ml ISD concentration

 

Extraction procedure:

Take 750µl of plasma and 500µl of internal standard, 250µl of Umeclidinium from the spiking solutions of both into a centrifuging tube and add 1ml of Acetonitrile go for cyclomixer for 15 sec. Then vertex for 2 min and finally centrifuge for 5 min at 3200rpm speed. After the centrifugation collect the sample and filter it directly inject 10µL into HPLC.

 

Optimized method:

Chromatographic conditions:

Mobile phase : 0.1% Ortho phosphoric acid pH (2.2): Acetonitrile (60:40)

Flow rate: 1.0ml/min

Column                 : BDS C18 (150mm x 4.6mm, 5m)

Detector wavelength: 215nm

Column temperature: 30⁰C

Injection volume: 10µL

 

Fig. 4:Chromatogram showing separated peaks of the drugs & IS in plasma.

 

Method Validation:

System suitability:

All the system suitability parameters were within the range and satisfactory as per US-FDA guidelines. The % CV for system suitability test was 0.33% for Retention time (RT) of Umeclidinium , 0.43% for Retention time (RT) and 0.42% for the area ratio (analyte area/IS area) of Umeclidinium, 0.31% for the area ratio (analyte area/IS area) of Vilanterol. The results were reported in table 1.

 

 

Table 1: System suitability results of selected drugs in human plasma along with IS

System Suitability
Analyte	Umeclinidium		Empagliflozin (IS)			Vilanterol		Empagliflozin (IS)
Sample Name	Analyte Area	Analyte RT (min)	ISTD Area	ISTD RT (min)	Area Ratio	Analyte Area	Analyte RT (min)	ISTD Area	ISTD RT (min)	Area Ratio
AQ MQC	591580	2.47	496612	3.303	1.1912	276278	3.30	496612	3.303	0.5563
AQ MQC	593379	2.48	493590	3.317	1.2022	276336	3.32	493590	3.317	0.5598
AQ MQC	589704	2.48	495620	3.318	1.1898	276380	3.32	495620	3.318	0.5576
AQ MQC	592305	2.49	497691	3.33	1.1901	276425	3.33	497691	3.33	0.5554
AQ MQC	596110	2.49	497582	3.335	1.1980	276486	3.34	497582	3.335	0.5557
AQ MQC	593743	2.50	497618	3.342	1.1932	276538	3.34	497618	3.342	0.5557
MEAN		2.484		3.324	1.19409		3.324		3.324	0.55677
SD		0.0082		0.0142	0.004974		0.0142		0.0142	0.001709
%CV		0.33		0.43	0.42		0.43		0.43	0.31

 

1. Selectivity/ Specificity:

To establish the selectivity of the method, possible interference at the retention time of Umeclidinium, Vilanterol and   Internal standard due to endogenous plasma components were checked during the validation. Selectivity was performed by testing six batches of K₂EDTA blank plasma and the mass detection of extracted blank plasma gave good selectivity of both drug and internal standard. No interferences were found at the retention times of analytes and internal standard.

 

2. Linearity:

Calibration was found to be linear over the concentration range of 1.25 to 50µg/ml for Umeclidinium, 0.25 to 10.0µg/ml Vilanterol. The coefficient correlation (r²) value was found consistently greater than 0.999 in all the cases. This indicating linearity of results and an excellent correlation between peak area ratios for each concentration of analytes.

 

A representative calibration curve is shown in fig. 5, which is obtained during the third precision and accuracy batch. Back calculated concentrations obtained for 3 calibration curves are summarized in the table 2 and 3.

 

Fig. 5: Linearity curves of Umeclidinium & Vilanterol

 

Table 2: Linearity results of Umeclidinium

Acquisition Batch ID	STD1	STD2	STD3	STD4	STD5	STD6	STD7	STD8
	Nominal Concentration (µg/mL)
	1.250	2.500	3.750	10.000	25.000	30.000	40.000	50.000
	Back Calculated Concentration (µg/mL)
P&A1	1.235	2.510	3.711	10.195	25.150	30.30	40.19	50.49
P&A2	1.238	2.520	3.815	10.100	25.548	30.29	40.20	51.50
P&A3	1.240	2.550	3.813	10.105	25.252	30.31	40.21	50.51
n	3	3	3	3	3	3	3	3
Mean	1.2377	2.5267	3.7797	10.1333	25.3167	30.3000	40.1983	50.8330
SD	0.00252	0.02082	0.05948	0.05346	0.20673	0.01000	0.00764	0.57773
%CV	0.20	0.82	1.57	0.53	0.82	0.03	0.02	1.14
% Mean Accuracy	99.01	101.07	100.79	101.33	101.27	101.00	100.50	101.67

 

Table 3: Linearity results of Vilanterol

Acquisition  Batch ID	STD1	STD2	STD3	STD4	STD5	STD6	STD7	STD8
	Nominal Concentration (µg/mL)
	0.250	0.500	0.750	2.000	5.000	6.000	8.000	10.000
	Back Calculated Concentration (µg/mL)
P&A1	0.253	0.508	0.754	1.99	4.99	6.10	7.89	9.87
P&A2	0.251	0.505	0.752	2.00	5.11	5.98	7.99	9.99
P&A3	0.249	0.498	0.761	2.01	5.03	5.99	8.00	10.10
n	3	3	3	3	3	3	3	3
Mean	0.2510	0.5037	0.7557	1.9993	5.0437	6.0233	7.9603	9.9863
SD	0.00200	0.00513	0.00473	0.00764	0.06263	0.06479	0.06389	0.11301
%CV	0.80	1.02	0.63	0.38	1.24	1.08	0.80	1.13
% Mean Accuracy	100.40	100.73	100.76	99.97	100.87	100.39	99.50	99.86

 

 

3. Precision and Accuracy:

The intraday and inter day accuracy and precision was assessed by analysing six replicates at five different QC levels like LLOQ, LQC, MQC and HQC. Accuracy and precision method performance was evaluated by determined by six replicate analyses for Umeclidinium at four concentration levels, i.e.,1.25µg/ml (LLOQ), 3.75 µg/ml (LQC), 25µg/ml (MQC) and 40.0µg/ml (HQC), Vilanterol at 0.25µg/ml (LLOQ), 0.75µg/ml (LQC), 5.0 µg/ml (MQC) and 8.0µg/ml (HQC),The intra-day and inter day accuracy of plasma samples were assessed and excellent mean % accuracy was obtained with range varied from 99.93% - 101.79%, and 99.72% - 101.84% for intraday and 100.20%-100.90 and 100.17%-101.58 for inter day respectively. The precision (%CV) of the analytes and plasma samples were calculated and found to be 0.02% - 3.17%  and 2.15% - 6.62% for intraday and 0.92% - 2.48% and 0.62% - 2.62% for inter day respectively. The results are summarized in table 4.

 

 

Table 4: Precision data of selected drugs in human plasma

Drug	Umeclidinium				Vilanterol
Acquisition Batch ID	HQC	MQC1	LQC	LLOQ QC	HQC	MQC1	LQC	LLOQ QC
	Nominal Concentration (µg/mL)				Nominal Concentration (µg/mL)
	40.000	25.000	3.750	1.250	8.000	5.000	0.750	0.250
	Back Calculated Concentration (µg/mL)				Back Calculated Concentration (µg/mL)
n	18	18	18	18	18	18	18	18
Mean	40.1947	25.5647	3.7725	1.2388	8.0107	5.0547	0.7550	0.2560
SD	0.00942	0.39837	0.04804	0.00970	0.02452	0.16749	0.00486	0.00906
%CV	0.02	1.56	1.27	0.78	0.31	3.31	0.64	3.54
% Mean Accuracy	100.49	102.26	100.60	99.11	100.13	101.09	100.67	102.40
Between Batch Precision and Accuracy					Between Batch Precision and Accuracy
n	18	18	18	18	18	18	18	18
Mean	40.1981	25.4594	3.7538	1.2466	8.0139	5.0368	0.7542	0.2539
SD	0.00751	0.29709	0.03286	0.02406	0.04988	0.12850	0.00736	0.00668
%CV	0.02	1.17	0.88	1.93	0.62	2.55	0.98	2.63
% Mean Accuracy	100.50	101.84	100.10	99.72	100.17	100.74	100.56	101.58

 

4. Recovery:

Recovery was determined by measuring the peak areas obtained from prepared plasma samples with those extracted blank plasma spiked with standards containing the same area with known amount of Umeclidinium and Vilanterol. The overall % mean recovery for Umeclidinium and Vilanterol was found to be 99.34% and 99.029%. The overall % mean recovery for Empagliflozin was found to be 99.64%.

 

5. Stabilities:

Long term stock solution stability for Umeclidinium: In bench-top stability, six replicates of LQC & HQC samples (3.75 and 40.0µg/ml) were analyzed for 9 hours at room temperature on the laboratory bench. The % mean stability was calculated and found to 99.68% for LQC and 100.49% for HQC respectively.

 

Long term stock solution stability for Vilanterol: In bench-top stability, six replicates of LQC & HQC samples (0.75 and 8.00µg/ml) were analyzed for 9 hours at room temperature on the laboratory bench. The % mean stability was calculated and found to 100.33% for LQC and 98.83% for HQC respectively.

Matrix samples stability at -28±5 °C for 37 days & -80±5 ⁰C

 

Long term stock solution stability for the Umeclidinium was determined at a concentration of LQC-HQC level after a storage period of 37 days at -28°C& -80°C in refrigerator. The % mean stability of the Umeclidinium was found to be 99.77%, 99.99%  at 28 ± 5°C and 96.22%, 99.99%  at 80 ± 5°C respectively.

 

Long term stock solution stability for the Vilanterol was determined at a concentration of LQC-HQC level after a storage period of 37 days at -28°C& -80°C in refrigerator. The % mean stability of the Vilanterol was found to be 99.80%, 100.19 at 28 ± 5°C and 101.35%, 99.95 at 80 ± 5°C respectively

 

CONCLUSION:

A simple, accurate, precise method was developed for the estimation of the Umeclidinium and Vilanterol in human plasma using the Empagliflozin as internal standard. Retention time of Umeclidinium and Vilanterol were found to be 4.83min and 7.88min. %CV of the Umeclidinium and Vilanterol were found to be 0.19% and 1.14%. %Recovery was obtained as 99.19% and 96.91%. The linearity concentration is in the range of 0.3-12µg/mL of Umeclidinium , 0.06-2.4µg/mL of Vilanterol. The lower limits of quantification were 0.3µg/mL of Umeclidinium and  0.06µg/mL of Vilanterol, which reach the level of both drugs possibly found in human plasma. Further, the reported method was validated as per the ICH guidelines and found to be well within the acceptable range.The proposed method is simple, rapid, accurate, precise, and appropriate for pharmacokinetic and therapeutic drug monitoring in the clinical laboratories.

 

CONFLICT OF INTEREST:

None.

 

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Accepted on 12.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1017-1022.