Int J App Pharm, Vol 14, Issue 2, 2022, 110-115Original Article

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF DEXMETHYLPHENIDATE AND SERDEXMETHYLPHENIDATE BY USING RP-HPLC IN BULK AND PHARMACEUTICAL DOSAGE FORM

SUNIL RAYUDU1, M. MANORANJANI2,*, D. RAMA SEKHARA REDDY3

1,3Department of Chemistry, Krishna University, Machilipatnam 521004, AP, India, 2Department of Chemistry, PB Siddhartha College of Arts and Science, Vijayawada 520010, AP, India
*Email: drmanoranjani@gmail.com

Received: 03 Nov 2021, Revised and Accepted: 15 Dec 2021

ABSTRACT

Objective: The current investigation was pointed at developing and progressively validating novel, simple, responsive and stable RP-HPLC method for the simultaneous measurement of active pharmaceutical ingredients of Dexmethylphenidate and Serdexmethylphenidate.

Methods: A simple, selective, validated and well-defined stability that shows isocratic RP-HPLC methodology for the simultaneous determination of Dexmethylphenidate and Serdexmethylphenidate. The chromatographic strategy utilized inertsil ODS column of dimensions 250x4.6 mm, 5 µ, using isocratic elution with a mobile phase of acetonitrile and 0.1% orthophosphoric acid (70:30). A flow rate of 1 ml/min and a detector wavelength of 262 nm utilizing the PDA detector were given in the instrumental settings. Recovery, specificity, linearity, accuracy, robustness, ruggedness were determined as a part of method validation and the results were found to be within the acceptable range. Validation of the proposed method was carried out according to an international conference on harmonization (ICH) guidelines.

Results: LOD and LOQ for the active ingredient were established with respect to test concentration. The calibration chart plotted was linear with a regression coefficient of R2>0.999, which means the linearity was within the limit.

Conclusion: The proposed method to be fast, simple, feasible and affordable in assay condition. During stability tests, it can be used for routine analysis of production samples and to verify the quality of drug samples during stability studies.

Keywords: Dexmethylphenidate, Serdexmethylphenidate, RP-HPLC, Development, Validation

© 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2022v14i2.43515. Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

Dexmethylphenidate, sold under the brand name Focalin among others, is a medication [1, 2] used to treat attention deficit hyperactivity disorder (ADHD) [3, 4] in those over the age of five years. If no benefit is seen after four weeks, it is reasonable to discontinue its use. It is taken by mouth [5]. The immediate-release formulation lasts up to five hours, while the extended-release formulation lasts up to twelve hours. Common side effects include abdominal pain [6, 7], loss of appetite [8], and fever [9, 10]. Serious side effects may include abuse [11], psychosis [12], sudden cardiac death [13, 14], mania [15], anaphylaxis [16], seizures [17], and dangerously prolonged erection. Safety during pregnancy and breastfeeding is unclear. Dexmethylphenidate is a central nervous system (CNS) stimulant [18]. How it works in ADHD is unclear. It is the more active enantiomer of methylphenidate.

Serdexmethylphenidate (SDX) is a prodrug of dexmethylphenidate created by the pharmaceutical company KemPharm. The compound was first approved by the FDA as one of the active ingredients in Azstarys for the treatment of Attention Deficit Hyperactivity Disorder (ADHD) in children, adolescents, and adults in March 2021. Co-formulation of SDX with dexmethylphenidate allows for a more rapid onset of action while still retaining up to 13 h of therapeutic efficacy. Due to the delayed onset and prolonged duration of effects following oral administration of SDX, several dosage forms containing SDX are currently under investigation for use as long-acting psychostimulant [19, 20] in the treatment of various CNS disorders [21], substance use disorder (SUD) [22, 23], and sleep disorders [24]. Under the developmental codename KP484, SDX is being investigated as part of a potential "super-extended duration" psychostimulant, with therapeutic efficacy lasting up to 16 h following oral administration. The aim of the study is to estimate the pharma ingredients Dexmethylphenidate and Serdexmethylphenidate by using RP-HPLC.

A B

Fig. 1: Structure of (A) Dexmethylphenidate and (B) Serdexmethylphenidate

Till today there is only one HPLC method [25] was reported in the literature. Hence we developed a method for the quantification of Dexmethylphenidate and serdexmethylphenidate. The developed HPLC method was utilized for the estimation of the drug by in vitro method.

MATERIALS AND METHODS

Chemicals

Acetonitrile (HPLC-grade), orthophosphoric acid, water were purchased from Merck India Ltd, Mumbai, India. APIs of Dexmethylphenidate and Serdexmethylphenidate standard was procured from Glenmark, Mumbai.

The instrumentation

Waters alliance liquid chromatography (model 2695) was monitored with empower 2.0 data handling system and a detector of photodiode array (model 2998) was used for this study [25, 26].

Method optimization

To optimize the chromatographic conditions, different ratios of phosphate buffer and the acetonitrile in the mobile phase with isocratic mode was tested. However, the mobile phase composition was modified at each trial to enhance the resolution and also to achieve acceptable retention time. Finally, 0.1% ortho phosphoric acid buffer and acetonitrile with isocractic elution was selected because it results in a greater response of active pharmacy ingredients. During the optimization of the method various stationary phases such as C8, C18 and amino, phenyl columns were tested. From these trials the peak shape was relatively good with inertsil ODS column of 250 x 4.6 mm, 5 µ with a PDA detector. The mobile phase flow rate has been done at 262 nm in order to obtain enough sensitivity. By using above conditions we get retention time of Dexmethylphenidate was about 4.535 min with a tailing factor of 1.24. The number of theoretical plates for Dexmethylphenidate was 7328 and Serdexmethylphenidate retention time was 2.936 min with a tailing of 1.24, plate count 3319 which indicate the column’s successful output the % RSD for six replicate injections were 0.25% for dexmethylphenidate and 0.25% for serdexmethylphenidate, the proposed approach suggests that it is extremely precise. According to ICH guidelines, the method established was validated.

Validation procedure

The analytical parameters such as system suitability, precision, specificity, accuracy, linearity, robustness, LOD, LOQ, forced degradation and stability were validated according to ICH Q2 (R1) guidelines [27-29].

Preparation of buffer

1 ml of orthophosphoric acid (OPA) is dissolved in 1 lt of HPLC grade water and filter through 0.45 µ filter paper.

Chromatographic conditions

The HPLC analysis was performed on a reverse-phase HPLC system with isocratic elution mode using a mobile phase of acetonitrile and 0.1% OPA and inertsil ODS (250x4.6 mm, 5 μ) column with a flow rate of 1 ml/min.

Diluent: Mobile phase was used as a diluent.

Preparation of the standard solution

For standard solution preparation, add 70 ml of diluents to 12 mg of Dexmethylphenidate and 56 mg of Serdexmethylphenidate taken in a 100 ml volumetric flask and sonicate for 10 min to fully dissolve the contents and then makeup to the mark with diluents (stock solution). Further, 5 ml of solution was drawn from the above normal stock solution into a 50 ml volumetric flask and diluted up to the level.

Preparation of sample solution

For sample solution preparation, add 70 ml of diluents to 118 mg of Dexmethylphenidate and Serdexmethylphenidate sample taken in a 100 ml volumetric flask and sonicate for 20 min to fully dissolve the contents and then makeup to the mark with diluents (stock solution). Further, 5 ml of solution was drawn from the above normal stock solution into a 50 ml volumetric flask and diluted up to the level.

RESULTS AND DISCUSSION

The main analytical challenge during the development of a new method was to separate active pharma ingredients. In order to provide good performance, the chromatographic conditions were optimized.

System suitability

In System suitability injecting standard solution and reported USP tailing and plate count values are tabulated in table 1 and the standard chromatogram was shown in fig. 2 [30].

Table 1: Results of system suitability

System suitability parameter Serdexmethylphenidate Dexmethylphenidate
Mean Std Dev % RSD Mean Std Dev % RSD
USP Plate Count 3319 43.614 1.31 7238 48.726 0.67
USP Tailing 1.15 0.008 0.66 1.03 0.008 0.79
USP Resolution - - - 7.53 0.036 0.48
Retention time 2.937 0.002 0.08 4.540 0.005 0.10

(n=6)

Fig. 2: Chromatogram of standard

Specificity

In this test method, placebo and standard solutions were analyzed individually to examine the interference. The below fig. shows that the active ingredients were well separated from blank and their excipients and there was no interference of placebo with the principal peak. Hence the method is specific [30].

Fig. 3: Chromatogram of blank

Linearity

The area of the linearity peak versus different concentrations has been evaluated for Serdexmethylphenidate as 25, 50, 75, 100, 125, 150 percent dilutions [30], respectively. Linearity was performed in the range of 14-84 µg/ml of Serdexmethylphenidate and 3-18 µg/ml of Dexmethylphenidate. The correlation coefficient achieved greater than 0.999 for all.

Accuracy

Three kinds of concentration levels of 50, 100, and 150 percent at a specified limit were used in this process to assess the accuracy of this particular method. The developed method was found to be highly accurate and reliable. The recovery percentages were given in table 3 [31].

Precision

In method, precision study prepare six different sample solutions in the concentration of Serdexmethylphenidate (56 µg/ml) and Dexmethylphenidate (12 µg/ml) were injected into HPLC system. The % assay results were found to be in the range of 98% to 102%. Peak areas were calculated, which were used to calculate mean, SD and % RSD values. These results are given below table 4 [32].

Table 2: Linearity results

S. No. Serdexmethylphenidate Dexmethylphenidate
Conc. µg/ml Serdexmethylphenidate area count Conc. µg/ml Dexmethylphenidate area count
1 14.00 1058753 3.00 285651
2 28.00 2271560 6.00 498128
3 42.00 3182175 9.00 751473
4 56.00 4366503 12.00 985033
5 70.00 5287600 15.00 1190570
6 84.00 6492881 18.00 1406743
Correl coef 0.99954 0.99905
Slope 76610.41 77583.00
intercept 19430.29 32838.43

A B

Fig. 4: Calibration plot of (A) Serdexmethylphenidate and (B) Dexmethylphenidate

Table 3: Results of accuracy

S. No. % Level Serdexmethylphenidate Dexmethylphenidate
Mean % recovery Std dev Mean % recovery Std dev
1 50 99.6 0.392 99.7 0.611
2 100 100.2 0.474 100.1 0.427
3 150 98.8 0.306 98.8 0.483

n=3

Table 4: Intraday precision results

S. No. Serdexmethylphenidate Dexmethylphenidate
Conc. (µg/ml) Area counts % Assay as is Conc. (µg/ml) Area counts % Assay as is
1 56 4362891 100.3 12 994314 100.2
2 4353789 100.1 990341 99.8
3 4335810 99.7 989739 99.7
4 4354608 100.2 996352 100.4
5 4359396 100.3 991343 99.9
6 4341312 99.8 990561 99.8
% RSD 0.26 0.27
mean 100.1 100.0
SD 0.258 0.273

n=6

Fig. 5: Chromatogram of method precision

Intermediate precision

Separate instruments were used on different days, in different locations, for independent investigations into six different replicates of the sample solution. Mean RSD values have been calculated and determined from the peak regions. The following table shows the results. Serdexmethylphenidate (56 μg/ml) and Dexmethylphenidate (12 μg/ml) were analysed on 6 different days with 6 different samples. Mean, standard deviation and percent related standard deviation values were calculated from peak areas. Thus, it has been found that the current method yields very accurate results, with RSD values less than 2 percent and percent assay values near 100 percent. In table 5 [33] we can see the results.

LOD and LOQ

The LOD concentration of serdexmethylphenidate was 1.68 µg/ml and s/n values is 5 and Dexmethylphenidate was 0.36 µg/ml and s/n values is 3. The LOQ concentration for Serdexmethylphenidate was 5.6 µg/ml and their s/n values are 26, and Dexmethylphenidate was 1.2 µg/ml and s/n values is 22. The method is validated as per the ICH guidelines [34, 35].

Robustness

The design of the experiment was intentionally altered in order to test the robustness of the system. Examples of such changes include changing the flow rate, organic to inorganic ratio, and so on. The results were robust and tabulated in table 7 [36].

Degradation studies

Partial degradation of the drug was accomplished using various forced degradation conditions on the Serdexmethylphenidate and Dexmethylphenidate sample. Research has been carried out to see if the method works for degrading products [37, 38]. Additionally, the studies describe the conditions under which the drug is unstable, providing further information so that appropriate precautions are taken during the process of formulation in order to avoid possible instabilities [39, 40].

Table 5: Inter-day outcomes of the accuracy of dexmethylphenidate

S. No. Serdexmethylphenidate Dexmethylphenidate
Conc.(µg/ml) Area counts % assay as is Conc. (µg/ml) Area counts % assay as is
1 56 4352889 100.1 12 994320 100.2
2 4353794 100.1 991346 99.9
3 4335815 99.7 991747 99.9
4 4354611 100.1 995359 100.3
5 4339397 99.8 990540 99.8
6 4341324 99.8 994556 100.2
% RSD 0.19 0.21
mean 99.9 100.1
SD 0.186 0.207

n=6

Table 6: LOD and LOQ for dexmethylphenidate

Serdexmethylphenidate Dexmethylphenidate
LOD LOQ LOD LOQ
Concentration s/n Concentration s/n Concentration s/n Concentration s/n
1.68µg/ml 5 5.6µg/ml 26 0.36µg/ml 3 1.2µg/ml 22

Table 7: Robustness data of dexmethylphenidate

Parameter name Serdexmethylphenidate Dexmethylphenidate
Mean SD %RSD Mean SD %RSD
Flow minus (0.8 ml/min) 99.7 0.252 0.25 99.7 0.205 0.21
Flow plus (1.2 ml/min) 99.5 0.265 0.27 99.9 0.379 0.38
Organic minus (63:37) 100.2 0.101 0.10 100.1 0.208 0.21
Organic plus (77:23) 100.1 0.265 0.26 99.9 0.265 0.27

RSD-Relative standard deviation; n=3

Acid degradation

1 ml of sample stock solution was moved to a volumetric flask of 10 ml, add 1 ml of 1N HCl and left it for 15 min. After 15 min add 1 ml of 1N NaOH and makeup to the diluent mark. Filter the solution using a syringe filter and injected it into HPLC system.

Alkali degradation

1 ml of sample stock solution was moved to a volumetric flask of 10 ml, add 1 ml of 1N NaOH and left it for 15 min. After 15 min add 1 ml of 1N HCl and make up to the mark. Filter the solution using syringe filter and injected into HPLC system.

Peroxide degradation

1 ml of sample stock solution was moved to a volumetric flask of 10 ml, add 1 ml of 30% hydrogen peroxide solution and make up to the mark with diluents. Filter the solution using syringe filter and injected it into HPLC system.

Reduction degradation

1 ml of sample stock solution was moved to a volumetric flask of 10 ml and add 1 ml of 30% sodium bi sulphate solution and makeup to the mark with diluents. Filter the solution using a syringe filter and injected into HPLC system.

Thermal degradation

The sample solution was set in an oven at 105 °C for 6 h. The resultant solution was injected into HPLC system.

Hydrolysis degradation

1 ml of sample stock solution was moved to a volumetric flask of 10 ml and add 1 ml of HPLC grade water and makeup to the mark with diluents. Filter the solution using syringe filter and injected it into HPLC system.

All degradation results are tabulated in table 9.

Table 9: Forced degradation results of dexmethylphenidate

Degradation condition Serdexmethylphenidate Dexmethylphenidate
% assay* % degradation* % assay* % degradation*
Acid degradation 87.8 12.2 89.0 11.0
Alkali degradation 87.9 12.8 88.6 11.4
Peroxide degradation 86.4 13.6 87.5 12.5
Reduction degradation 88.6 11.4 80.6 9.4
Thermal degradation 97.9 2.1 98.1 1.9
Hydrolysis degradation 98.3 1.7 98.9 1.1

*Data expressed as mean; n=3

CONCLUSION

The developed method was accurate, precise and reliable for the simultaneous analysis of Serdexmethylphenidate and Dexmethylphenidate in pharmaceutical formulations. This method was validated for linearity, accuracy, precision, robustness, forced degradation of Serdexmethylphenidate and Dexmethylphenidate. The RSD values for all parameters were found to be less 2, which indicates the validity of the method and results obtained by this method are in fair agreement. Finally, this method can be used for better analysis of Serdexmethylphenidate and Dexmethylphenidate.

ACKNOWLEDGEMENT

The authors are thankful to Shree Icon Pharmaceutical Laboratories to complete this research work.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All authors have contributed equally.

CONFLICT OF INTERESTS

Declared none

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