Method development and validation of droxidopa by HPLC technique

Thepurpose of thiswork is to develop and validate stability, indicating reverse phase High-performance liquid chromatography (HPLC)method for the rapid and precise determination of droxidopa in its pure form and formulations. A simple, fast, accurate and economical way has been developed and validated for the quanti ication of droxidopa by HPLC technique. The chromato graphic system was equipped with Shimpack columnC18((250x 4.6) mm, 5μ) as stationary phase and UV detector at 220 nm, in conjunction with a mobile phase of phosphate buffer pH 2.0:acetonitrile (60:40,% v/v) at a low rate of 1.0 mL/min. The developed HPLC technique was found to be rapid as the retention time was 2.2 minutes for droxidopa peak to elute. The method was validated as per the International Conference on Harmonization (ICH) guidelines for speci icity linearity, accuracy, precision. The developedmethod was selective with well-resolved peak. Linearity was observed over the concentration range of 25-150μg/mL for droxidopa. The recovery of Droxidopa was found to be 100.54% 101.65%. Statistical techniques were employed for the validation of precision, linearity, accuracy, robustness and ruggedness and can be applied for routine analysis. Validation revealed that the developed method was speci ic, accurate, precise, reliable, robust, reproducible and suitable for the systematic quantitative review.


INTRODUCTION
Orthostatic hypotension is a signi icant problem which affects skeletal-motor actions, multiple system degeneration as well as in Parkinson's disease. Treatment using pharmacological measures in maintaining a suf icient amount of the noradrenaline is not just enough because the damage to nerve terminals or ganglia or central autonomic networks occurs. A preferred way is prescribing sympathomimetics along with the other pharmacological agents that affect physiological factors which help to reduce blood pressure. Droxidopa is chemically L-threo-dihydroxyphenylserine (Figure 1 ), is a pro-drug, which differs from noradrenaline by a carboxyl group. It can be allowed to take orally, unlike noradrenaline (Mathias, 2008). Droxidopa is a pro-drug of noradrenaline, which escalates the amounts of neurotransmitters in the human body and brain (Goldstein, 2006).
A literature survey reveals that only one method based on HPLC (Ankit et al., 2017) is available for determination of droxidopa in the formulation and HPLC and LC-MS/MS (Chen and Hewitt, 2018;Derangula et al., 2018;Wang et al., 2016) methods have been reported for estimation of drug and its metabolites in biological luids. These methods are complicated, costly, and time-consuming with complicated mobile phases and gradient elution, leading to baseline shifting in comparison to a simple HPLC-UV process. Overall the presented method was to establish speci ic, robust, accurate, optimised and validated RP-HPLC technique which can be applied for the quanti ication of droxidopa in the presence of degradants. The optimised isocratic HPLC technique was validated cohering to the ICH guidelines (Ich, 2005).

Chemicals and Reagents
Droxidopa was obtained as a gift sample from the reputed pharmaceutical organisation. Potassium dihydrogen phosphate, orthophosphoric acid, HPLC grade acetonitrile, water for HPLC was sourced from Rankem, India.

Preparation of phosphate buffer
Potassium dihydrogen phosphate (0.01 mM)in 990mL of HPLC water and pH ixed at 2.0 using orthophosphoric acid and made up to 1000mL with water, Filter the buffer solution.

Wavelength Selection
From the standard stock solution, 1µg/mL of the solution was prepared in mobile phase by diluting 1mL of the stock solution to 10 mL. After preparing, the solution was exposed to UV (Shimadzu-1700) for the scrutinisation of wavelength between 200-400nm. The λ max of the drug droxidopa was found to be at 220nm (Figure 2 ).

Chromatographic separation
Chromatographic separation was obtained with phosphate buffer: acetonitrile in the proportion of 60:40 %v/v with a Shimpack column C 18 (250 mm x 4.6 mm, 5µ) and asolvent speed of 1.0 mL/min., injection capacity of 10 µL, ambient column temperature and detection at a wavelength of 220nm by PDA detector.

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

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

Procedure for method validation
Validation of the proposed HPLC method was carried out as per the ICH guidelines Q2 (R1) for precision, linearity, accuracy, robustness and ruggedness (Guideline, 2005). To establish an HPLC testing system meets the requirement for the purpose; it was veri ied by injecting six replicates of standard solution and various parameters like tailing factor, theoretical plates were evaluated statistically.

Speci icity
Speci icityin the method is the competence to measure the analyte in the presence of its excipients and degradation products. Commonly used formulation excipients like cellulose derivatives, starch, sucrosebased polyvinyl pyrrolidone, polyethene glycol and lactose were spiked into a pre-weighed quantity of droxidopa; appropriate dilutions made and tested how well the method can recognise the analyte.

Forced Degradation Studies
Forced Degradation Studies of droxidopa, was performed atvarious stress conditions as speci ied in ICH guideline Q1A (R2) (ICH, 2005). The droxidopa solution containing100µg/ml was exposed to the acidic, alkaline, oxidative, thermal condition. Acidic and alkaline degradation was performed at 80ºC temperature and neutralised before injecting. Oxidative stress studies were carried out for using 3% H 2 O 2 (Reynolds et al., 2002).The degraded samples, along with droxidopa control sample, were chromatographed and examined for any interferences at the retention time of the droxidopa peak from the potential degradants.

Precision
The precision was assessed by performing repeatability at a target drug concentration of 100µg/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 a concentration from 25 to 150µg/mL were prepared and diluted with the mobile phase from the standard stock solution. Calibration curve designed by plotting peak area against concentrations of droxidopa, correlation coef icient calculated.

Limit of detection and limit of quanti ication
LOD is the smallest concentration of the analyte which involves an estimable response (Signal to noise ratio 3) whereas LOQ is the level at which precision is weaker than a speci ic value (RSD ≥3.0% or Signal to noise ratio 10)

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 con irmed by recovery experiments. The accuracy was assessed by triplicate determinations of three different solutions having 160,200 and 240 µg/mL.

HPLC method development and optimisation
To attain the best separation conditions, the optimisation of the mobile phase was to provide desirable selectivity and sensitivity in low run time. Columns from different brands were evaluated. Eventually, a symmetrical peak of the analyte with a justi iable low run time was ful illed by engaging buffer and acetonitrile, and Shimpack column C 18 (250 mm x 4.6 mm, 5µ)was inalised, as it furnished better peak symmetry about 1.25,theoretical plates and irm baseline. The validation variables are linearity, precision accuracy, robustness for the inalised chromato graphic parameters of the HPLC method of droxidopain solid dosage form and quanti ication for bulk and dosage forms.

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. The acceptance criterion for theoretical plates was not less than 4000. The system suitability parameters inalised for the analysis of droxidopaare in Table 1.
The speci icity trial displayed that pharmaceutical additives and degradation products did not intrude with the peak of the droxidopa. None of the peaks was eluted at the retention time of droxidopa( Figure 3 ). This proves that the proposed method was particular for quantitation of droxidopa in the formulation (Shabir, 2003). The results of the degradation studies using different optimised condition are listed in Table 2.

Linearity
Acceptable linearity range was illustrated in the present work for droxidopa over the range of 50-150 µg/mL (Jain, 2009). A calibration curve was plotted for ive standard concentrations of droxidopa versus droxidopa peak area, a regression equation was computed, and the linearity of the calibration curve (Figure 4 ) and linearity overlap ( Figure 5 ) was established by the high value of correlation coef icient,r 2 : 0.999justi ies the outstanding correlation linking the concentrations and peak area of droxidopa and are briefed in Table 3. The detection limit and quanti ication limit were obtained from the slope of the linear regression curve. The limit of detection is 2.521µg/mL, and the quanti ication limit is found to be 7.534 µg/mL.

Accuracy studies
Accuracy was accomplished at three levels (80%, 100% and 120%). Triplicate analyses carried out at 160, 200 and 240 µg/mL and average recoveries were studied and shown in Table 4.

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 was100.83 with RSD of 0.303. % RSD<2.0 indicates that the method is precise (Sabir et al., 2013). The ruggedness was veri ied by performing the chromatographic analyses of samples by various analysts on two different days. Results are presented in Table 5.

Robustness
There were nonoticeable changes in the chromatographic pattern when deliberate changes were made in the chromatographic conditions, thus making the method robust.

CONCLUSIONS
A fast, sensitive, precise, rugged, accurate RP-HPLC technique was progressed for droxidopa, and the evolved method was validated as per ICH Q2 (R1A) guidelines. The % RSD of < 2.50 % shows the HPLC technique to be precise. Excellent linearity of droxidopa is shown from 25 to 150 µg/ml. Recovery experiments that were accomplished exhibited the accuracy of the method. This ranged between 100.54% -101.65%. Thus it can be accomplished that an accurate, precise and rugged RP-HPLC technique has been optimised and validated for the regular estimation of droxidopa in pure and oral dosage formulations.