INTRODUCTION:

Orthostatic hypotension is a major problem which effects skeletomotor actions, multiple system degeneration as well as in Parkinson's disease. Treatment using pharmacological measures in maintaining adequate amounts of the neurotransmitter noradrenaline is not successful because of damage to nerve terminals or ganglia or central autonomic networks. A preferred way is prescribing sympathomimetics in addition to other pharmacological agents that target physiological mechanisms that contribute to blood pressure control. Droxidopa is chemically L-threo-dihydroxyphenylserine, is a pro-drug which is structurally similair to noradrenaline, but with a carboxyl group. It can be administered orally, unlike noradrenaline [1]. Droxidopa as a prodrug of noradrenolone, which increase the amounts of neurotransmitters in the body and brain [2].

Detailed literature survey for droxidopa disclosed that, there is no reported UPLC method for quantification of Droxidopa. Ankit B. Chaudhary has developed a method by HPLC for estimation of droxidopa [3].

Kumar et al. has developed and validated HPLC method to study the degradation behavior and related impurities of Droxidopa [4]. Venkata Ramu Derangula et al [5] has reported a LC-MS/MS method for estimation of droxidopa in biological fluids, these methods are complicated, costly, and time-consuming in comparison to a simple UPLC method.

Figure 1: Chemical Structure of Droxidopa.

Moreover, the HPLC method reported by Ankit B. Chaudhary et al., (3) for the determination of content of droxidopa in solid dosage form shows longer run times, droxidopa peak at 3.6 min, as compared to the developed method and consequently consumes more amounts of organic solvents, which may have negative effect on the environment, therefore resulting in expensive disposal process. Since there is no reported UPLC method for estimation of droxidopa, these instigated the authors to attempt and develop a UPLC method which is specific, reliable and economical. The present work depicts the development of a UPLC method and validation of the same in the presence of pharmaceutical additives according to the ICH guidelines (ICH, 2005). The developed method can be put into practical use for the regular assessment of droxidopa in dosage forms.

EXPERIMENTAL:

Chemicals and Reagents:

Droxidopa was obtained as a gift sample. Triethyl amine, ortho phosphoric acid, HPLC grade methanol, water for HPLC was sourced from Rankem, India.

Preparation of triethylamine buffer:

By mixing 1mL of Triethyl amine in 990mL of HPLC water and pH fixed at 3.0 using ortho phosphoric acid and made up to 1000mL with water, Filter the buffer solution.

Mobile phase composition:

Tri ethyl amine buffer: methanol (25:75 %v/v), 250mL of buffer and 750mL of HPLC grade methanol mixed.

Instrumentation:

Analytical separation was achieved on Ultra Performance Liquid chromatography (Agilent 1220 Infinity with Open Lab CDS chemstation) in isocratic mode.

Chromatographic separation:

Chromatographic separation was obtained with triethylamine buffer: methanol in the proportion of 25:75 %v/v with a Phenomenex column C18 (50mm x 3.00 mm, 3µ) and a solvent speed of 1.0mL/min., injection capacity of 2µL, ambient column temperature and detection at a wavelength of 235nm by UV detector.

Preparation of Droxidopa standard solution (200µg/mL):

Droxidopa working standard, 25mg accurately weighed, dissolved in 5mL of 0.1N HCl and diluted with a suitable volume of mobile phase to 25mL (1000 ppm). The standard stock solution was diluted with a suitable volume of mobile phase to get 200ppm.

Sample solution:

Droxidopa sample powder equivalent to about 25mg, exactly weighed and dissolved in 5mL of 0.1N hydrochloric acid and the volume was made up to 25mL with a mobile phase to produce a solution of 1000ppm. The sample stock solution was diluted with a suitable volume of mobile phase to get 200ppm.

Procedure for method validation:

Validation of the proposed UPLC method was carried out as per the ICH guidelines Q2 (R1) [6] for precision, linearity, accuracy, robustness and ruggedness.

Specificity:

Specificity in a method is the competence to measure the analyte in the presence of its excipients. Commonly used formulation excipients like cellulose derivatives, starch, sucrose, polyvinyl pyrrolidone, polyethylene glycol and lactose were spiked into a pre weighed quantity of droxidopa; appropriate dilutions made and tested how well the method can recognize the analyte.

System suitability:

To establish UPLC testing system meets the requirement for the purpose, it was verified by injecting six replicates standard solution and various parameters like tailing factor, theoretical plates were evaluated statistically.

Precision:

The precision was assessed by performing repeatability at target drug concentration of 200μg/mL in one day and % RSD was calculated. The precision studies were also repeated on subsequent days to determine intermediate precision and by different analysts.

Linearity and range:

Five levels of calibration solutions at concentration from 100 to 300μg/mL were prepared and diluted with the mobile phase from standard stock solution. Calibration curve constructed by plotting peak area against concentrations of droxidopa, correlation coefficient calculated.

Limit of detection and limit of quantification:

Limit of detection is the smallest concentration of the analyte which involves an estimable response (signal to noise ratio 3) whereas Limit of quantification is the level at which precision is poorer than certain value (RSD ≥ 3.0% or Signal to noise ratio 10)

LOD = 3.3 x σ/S

LOQ = 10 x σ/S

Where, σ is the standard deviation of y-intercepts of regression lines and S is the slope of the calibration curve.

Accuracy:

Method accuracy confirmed by recovery experiments. The accuracy was assessed by triplicate determinations of three different solutions having 160, 200 and 240 µg/mL with in- house mixture of placebo.

Robustness:

Robustness of the evolved UPLC method was carried out by intentionally changing the chromatographic conditions. The mobile phase flow rate (1±0.1mL/min.), buffer pH (3.0±0.2) and wavelength (235± 2) were changed. The % RSD of the theoretical plates, tailing factor and retention time was compared with those obtained under the nominal UPLC conditions.

RESULTS AND DISCUSSION:

UPLC method development and optimization:

To attain the best separation conditions, optimization of the mobile phase was to provide desirable selectivity and sensitivity in a low run time [7]. The usage of methanol and addition of peak modifiers resulted in superior column efficiency and lesser run time. Columns from different brands were evaluated, and Phenomenex column C₁₈ (50mm x 3.0mm, 3µ) was finalized, as it furnished better peak symmetry about 1.25, theoretical plates and firm baseline. The validation variables are linearity, precision accuracy, robustness for the finalized chromatographic parameters of the UPLC method of droxidopa in solid dosage form and quantification for bulk and dosage forms. Fig. 4 shows a representative chromatogram of droxidopa by proposed UPLC method.

METHOD VALIDATION:

System suitability:

The peak symmetry and theoretical plates were calculated for the working solutions. The acceptance criteria for peak area counts should be not more than 2.0 % RSD and for tailing factor not more than 2.0 for the analyte peak [8]. The acceptance criterion for theoretical plates was not less than 500. The system suitability parameters finalized for the analysis of droxidopa are in table 1.

Table 1: System Suitability studies.

Name	Retention time	Peak area ± SD	Theoretical plates ±SD	Asymmetry ±SD
Droxidopa	0.32 min.	71.163±0.44	641.42 ± 6.16	1.33 ± 0.01

Specificity:

The specificity trial displayed that pharmaceutical additives did not intrude with the peak of the droxidopa. None of the peaks were eluted at the retention time of droxidopa as shown in figure 3. This proves that the proposed method was particular for quantitation of droxidopa in the formulation.

Linearity:

Acceptable linearity range was illustrated in the present work for droxidopa over the range of 100–300µg/mL [9]. A calibration curve was planned for five different concentrations of drug versus corresponding peak area, regression equation was computed (figure 2), y = 0.3409x ± 654. The linearity of the calibration curve was established by the high value of correlation coefficient, r2: 0.999 justifies the outstanding correlation linking the concentrations and peak area of droxidopa and are briefed in table 2.

Sensitivity:

The detection limit and quantification limit were obtained from slope of linear regression curve. The limit of detection is 2.5µg/mL and quantification limit is found to be 7.5µg/mL (Table 2).

Table 2: Regression characteristics determined by the proposed method

Parameters	Droxidopa
LOD	2.5 µg/mL
LOQ	7.5 µg/mL
Linearity range (µg/mL)	100-300µg/mL
Slope (b)	0.3409
Intercept (a)	0.654
Correlation coefficient (r)	0.999

Figure. 2: Linearity graph for droxidopa

Accuracy studies:

Accuracy was accomplished at three levels (80%, 100% and 120%). Triplicate analyses carried out at 160, 200 and 240µg/mL and average recoveries were calculated and shown in table 3.

Method precision and Intermediate precision:

The precision of the method was carried by performing six independent determinations. The average assay of six determinations of droxidopa was 99.75 with RSD of 1.04%. % RSD < 2.0 indicates that the method is precise. The ruggedness was verified by performing the chromatographic analyses of samples by various analysts on two different days. Results are presented in Table 4.

Robustness:

The developed method was verified for robustness by leading about small changes in the exploratory surroundings chromatographic conditions, pH of the buffer, flow rate, and detection at a different wavelength [10]. There were no significant changes in the chromatographic pattern when the modifications were made in the experimental conditions, thus showing the method to be robust as shown in table 5.

Table 4: Ruggedness

Sl. No.	Analyst-1 (% Assay)	Analyst-2 (% Assay)
1	100.93	99.22
2	100.91	98.17
3	101.00	100.42
4	100.76	98.67
5	101.77	99.39
6	100.68	99.15
Mean (n=6)	99.75	99.70
SD (n=6)	1.04	1.05
%RSD (n=6)	1.04	1.05
Overall mean (n=12)	100.09
Overall SD (n=12)	1.12
Overall %RSD (n=12)	1.12

Table 5: Chromatographic conditions investigated during robustness study.

	Retention time (RSD ≤ 2.0, n=6)	Tailing factor (RSD ≤2.0, n=6)	Mean Peak area (% RSD ≤2.0, n=6)
0.9 mL/min.	1.46	1.40	72.54 (0.38)
1.1 mL/min.	1.80	1.03	70.62 (0.43)
At wave length 233 nm	1.73	1.18	76.65 (0.85)
At wave length 237 nm	1.73	1.48	71.72 (0.31)
At 2.8 pH	1.46	1.40	71.47 (0.28)
At 3.2 pH	1.48	1.03	70.62 (0.43)

CONCLUSION:

Accounting for the efficiency of UPLC, trial has been made to develop simple, accurate, precise, speedy and economic UPLC method for estimation of Droxidopa. The benefits of the developed method are linear, accurate, precise, reproducible, simple and economical also because of affordable reagents and less run time. The described method purely offers a rapid determination of droxidopa peak at 0.35 min. and 1.5 minutes run time, the drug showed good linearity over the range of 100-300μg/ml of concentration of droxidopa with co-efficient of correlation, (r²) 0.999. The result of the recovery analysis of solid dosage form by the recommended method is highly reproducible. The UPLC method can be incorporated for the usual quality control analysis of the Droxidopa in dosage form.

Figure 3: Typical UPLC chromatogram of placebo solution

Figure 4: Typical UPLC chromatogram of droxidopa solution (200µg/mL)

ACKNOWLEDGEMENTS:

The authors are thankful to Synthiya Research Labs Pvt. Ltd, Pondicherry, India for providing necessary chemicals; equipment and facility to complete research work.

REFERENCES:

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3. Ankit B. Chaudhary, Ritu D. Patel, Monica J. Hingu. Analytical method development and validation of droxidopa. WJPPS 2017;6(10): 921-932

4. Jack J. Chen L. Arthur Hewitt. Comparison of the pharmacokinetics of Droxidopa after dosing in the fed versus fasted state and with 3-times-daily dosing in healthy elderly subjects. Drugs R D. 2018; 18: 77–86.

5. Venkata Ramu Derangula, Jyothi Thumma, Venkateswarlu Ponneri. Development and validation of LC-MS/MS method for the estimation of droxidopa in human plasma. Int Journal Chem Tech. 2018;11(11); 232–241

6. Validation of Analytical Procedures: Text and Methodology (Q2 (R1). ICH, 2005.

7. Swartz M.E. UPLC: An Introduction and Review. Waters Corporation, Milford, USA. 1259-1260.

8. Jane Jacob, Sreekanth Nadig. A UPLC method for simultaneous estimation of emtricitabine, tenofovir disoproxil fumarate and efavirenz in pharmaceutical dosage forms. Research J. Pharm. and Tech 2017; 10(12): 4463-4466.

9. S. Madhavi, A. Prameela Rani. Development and validation of RP-UPLC method for simultaneous estimation of Cobicistat and Darunavir. Research J. Pharm. and Tech 2017; 10(12): 4343-4349

10. Sri Datla V.V. S. S. N. Raju, A. Manikandan, S. Venkat Rao. Validation of Simple Isocratic RP-UPLC Method for Glecaprevir and Pibrentasvir determination and its Application in the Study of Stress Degradation. Research J. Pharm. and Tech 2019; 12(9): 4299-4304.

Received on 23.04.2020 Modified on 29.05.2020

Research J. Pharm. and Tech. 2021; 14(4):2125-2128.

DOI: 10.52711/0974-360X.2021.00376

Drug	Accuracy level	Amount of drug (µg/mL)	Quantity added (µg/mL)	Recovered (μg/mL) ± SD (n=3)	% Accuracy ± SD (n=3)
Droxidopa	Level-1	100	60	160.27±0.802	100.17±0.501
	Level-2	100	100	201.28±0.330	100.63±0.165
	Level-3	100	140	241.82±0.476	100.07±0.183