Development and validation of high performance liquid chromatography method for the determination of nifedipine in drug - excipient compatibility testing samples

To develop and validate the determination method of nifedipine in drug - excipient compatibility testing samples by high performance liquid chromatography.

DEVELOPMENT AND VALIDATION OF HIGH PERFORMANCE

LIQUID CHROMATOGRAPHY METHOD FOR THE

DETERMINATION OF NIFEDIPINE IN DRUG - EXCIPIENT

COMPATIBILITY TESTING SAMPLES

Tran Quang Trung 1 ; Nguyen Thanh Hai 2 ; Trinh Van Lau 3 ; Truong Ngoc Hien 1

SUMMARY

Objectives: To develop and validate the determination method of nifedipine in drug - excipient compatibility testing samples by high performance liquid chromatography Methods: Chromatographic conditions include: PDA detector, analytical wavelength, column and mobile phase were studied; the evaluation of the system suitability, specificity - selectivity, the linearity range, repeatability, intermediate precision, accuracy, range, limit of detection and limit of quantitation following the guidance of ICH Results: Chromatographic conditions were established, including: PDA detector, analytical wavelength 237 nm, column RP-C18 Phenomenex, mobile phase MeOH:H 2 O (65/35, v/v) The method had been evaluated and achieved the ICH’s regulations Conclusion: The developed HPLC method can be used to determine the nifedipine in drug - excipient compatibility testing samples, which will use in pre-formulation studies of the nifedipine dosage forms

* Keywords: Nifedipine; High performance liquid chromatography; Drug - excipient compatibility

INTRODUCTION

Nifedipine (NIF), a dihydropyridine

calcium channel blocker, is widely used in

the treatment of angina, hypertension and

other vascular disorders [2, 3] However,

nifedipine is relatively unstable [4] and

can interact with excipients in tablets

formulation that reduce its content In

order to determine the interaction between NIF and excipients to select suitable excipients for formulation development of NIF push - pull osmotic pump extended-released tablets, we have developed and validated the HPLC method to quantify NIF in drug - excipient compatibility testing samples created by combining NIF with potential excipients

1 Vietnam Military Medical University

2 Vietnam National University, Hanoi

3 National Institute of Drug Quality Control

Corresponding author: Tran Quang Trung (tqt201316@gmail.com)

Date received: 05/10/2019

Date accepted: 21/11/2019

MATERIALS AND METHODS

1 Materials and equipment

* Materials and chemicals:

- Reference standard of NIF (potency

99.82%) was provided by National Institute

of Drug Quality Control

- NIF was supplied by Baoji Guokang

Bio-Technology Co., Ltd (China) and

meet USP 38 standards

- PEO (polyethylene oxide) N10, PEO

N80, PEO N750, PEO 301, PEO 303,

PEO Coagulant provided by Colorcon

Asia Pacific Pte., Ltd

- Lactose monohydrate, sodium chloride,

magnesium stearate, PVP K30, red iron

oxide were sourced from China and all

meet USP 38 standards

- HPLC grade acetonitrile, methanol

were procured from Merck Ltd

- Other used chemicals were of HPLC

or analytical grade

* Equipment:

HPLC system Alliance Waters 2695D;

Detector 2998 dual α Absorbance (USA);

Phenomenex LUNA column, Xbridge

column (RP-C18, 5 µm, 250 x 4.6 mm);

Spectrometer EMC-61PC-UV, Emclab

(Germany); pH meter Mettler Toledo

(Switzerland); analytical balance Mettler

Toledo (precision 0.1 mg), other usually

equipment in laboratory

2 Methods

* Method development:

- Preparation for standard solutions:

+ Standard stock solution: A standard

stock solution containing 500 µg/mL NIF

was prepared by exactly approximately

weighing 50.0 mg NIF standard,

transferring it into a 100 mL volumetric flask and adding about 70 mL mobile phase composed of methanol/H2O (65/35, v/v) The flask was sonicated for

30 mins, allowed to cool at room temperature and the volume was made

up with mobile phase

+ Standard solution: Accurately taken

3 mL of the stock standard solution

500 µg/mL into a 10 mL volumetric flask and made up the volume with mobile phase, mixed well to give a standard solution of about 150 µg/mL of NIF All standard solutions were filtered through a 0.45 µm PTFE membrane filter prior to injection into the HPLC system

- Preparation for sample solutions:

Accurately weighed an amount of powder mixture of NIF and excipients (in

a ratio of 1/1) equivalent to about 50.0 mg

of NIF, transfered into a 100 mL volumetric flask; 70 mL mobile phase (methanol/H2O, 65/35, v/v) was added, and the flask was sonicated for 30 mins, allowed to cool at room temperature and made up the volume with mobile phase From this solution, a volume of about 10 mL of solution was withdrawn and was centrifuged at 4,000 rpm for 20 minutes Taken exactly 3 mL

of centrifuged solution into a 10 mL volumetric flask and add the mobile phase just to the mark, mixed well, filtered through a 0.45 µm PTFE membrane filter prior to injection into the HPLC system

- Chromatographic conditions:

The NIF standard solution of 150 µg/mL concentration was used for investigation

of chromatographic conditions The solutions were scanned under 200 - 500 nm wavelength range to find the maximum

absorption wavelength The other

chromatographic conditions such as:

stationary phase (silica gel column C18

Phenomenex LUNA, Xbridge); mobile

phase (mixture of solvents MeOH, ACN,

H2O at different proportions) were also

studied Maintained a flow rate at

1 mL/min, sample injection volume of

25 µL Identified chromatographic

conditions for peaks with good symmetry,

max peak area/concentration ratio and

peak height/concentration ratio

* Method validation:

The proposed HPLC-UV method was validated in accordance with the International Conference on Harmonization (ICH) guidelines and Circular 32/2018 of the Vietnamese Ministry of Health [1,5], by evaluating the validation characteristics such as the system suitability, specificity - selectivity, the linearity range, precision (repeatability, intermediate precision), accuracy, specified range, limit of detection and limit of quantitation

RESULTS AND DISSCUSION

1 Method development results

* Analytical wavelength:

The appropriate wavelength for NIF analysis was determined by recording UV spectrum of a NIF standard sample of 150 µg/mL on the EMC-61PC-UV spectrophotometer, Emclab The solution above was scanned from 200 to 500 nm with quartz cuvettes 1 cm of thick The blank sample was mobile phase composed of methanol/H2O (65/35, v/v)

Figure 1: Ultraviolet absorption spectrum of NIF standard solution of 150 µg/mL

UV-Vis spectrum showed that the absorption maxima of NIF was 237 nm and 370 nm From there, choose the wavelength 237 nm as the wavelength for NIF qualification by HPLC method

* Column efficiency validation:

Analyzed samples on two reversed silicagel columns C18 Xbridge and Phenomenex LUNA

Table 1: Column analysis validation

Results

Figure 2: Chromatogram of the NIF standard sample using Xbridge column

Figure 3: Chromatogram of the NIF standard sample using

Phenomenex LUNA column

The results of asymmetry factor, the number of theoretical plates, retention time and

chromatograms showed that both columns had a symetrical peak, the AF was within the permitted limits (0.8 ≤ AF ≤ 1.5); however, the Phenomenex LUNA column with a smaller number of theoretical plates, shorter retention time compared to these ones in the Xbridge column (13.108 mins), therefore, the Phenomenex LUNA column was selected for the quantitative analysis of NIF

* Mobile phase:

Based on the reference documents and chemicals, solvents available in laboratory, three different mobile phase systems were studied including:

- System 1: MeOH:H2O (65/35, v/v)

- System 2: ACN:H2O (65/35, v/v)

- System 3: ACN:MeOH:H2O (25/25/50, v/v/v)

Table 2: Mobile phase investigation results

Figure 4: Chromatogram of standard sample in mobile phase system MeOH:H2O

(65/35, v/v)

Figure 5: Chromatogram of standard sample in mobile phase system ACN:H2O

(65/35, v/v)

Figure 6: Chromatogram of standard sample in mobile phase system ACN:MeOH:H2O

(25/25/50, v/v)

Chromatogram of standard sample in system 2 included ACN:H2O (65/35, v/v) had unbalanced peak with AF = 1.261 and the baseline was not stable That one in system

3 composed of ACN:MeOH:H2O (25/25/50, v/v/v) had balanced peak but retention time was too short (1.050 minutes) Whereas, this one in system 1 including MeOH:H2O (65/35, v/v) had stable baseline, sharp and proportioned peak with AF = 1.126, indicated good separation ability

* Mobile phase ratio:

The mobile phase system which composed of mixture of MeOH and H2O was investigated at different ratio 50/50, 65/35, 60/40, 70/30, 80/20

Table 3: Results of investigation mobile phase ratio

Figure 7: NIF standard chromatogram with mobile phase MeOH:H2O (50:50, v/v)

Figure 8: NIF standard chromatogram with mobile phase MeOH:H2O (65:35, v/v)

Figure 9: NIF standard chromatogram with mobile phase MeOH:H2O (60:40, v/v)

Figure 10: NIF standard chromatogram with mobile phase MeOH:H2O (70:30, v/v)

Figure 11: NIF standard chromatogram with mobile phase MeOH:H2O (80:20, v/v)

It was found that the mobile phase with the ratio of MeOH:H2O (65:35, v/v) was

selected for quantitative analysis of NIF, because of good separation with a suitable

retention time, compact and balanced peak

2 Method validation results

* System suitability test:

Replicated the injection of a NIF standard solution at concentration of about

150 µg/mL 6 times on HPLC system Evaluated retention time (RT), peak area and

asymmetry factor and theoretical plate (N) in all chromatograms

Table 4: Results of investigation system suitability

The results showed that %RSD for the retention time, peak area, asymmetry factor

and theoretical plate of NIF standard were less than 2.0% The obtained value

demonstrated the suitability of the system for the analysis of NIF

* Specificity:

Prepared 3 samples: blank sample (mixture of excipients), standard sample, test

sample Sample processing and chromatography carried out under selected conditions

Figure 12: Chromatogram of blank sample

Figure 13: Chromatogram of NIF standard solution at concentration of 150 µg/mL

Figure 14: Chromatogram of NIF test sample at concentration of 150 µg/mL

Standard sample peak was balanced, retention time was 8.874 minutes On the

chromatogram of the blank sample, no peak was observed in a period of 8 - 10

minutes The chromatograms of both the test sample and the standard sample had

1 peak at approximately 8.8 minutes From there, we concluded that the method was

highly specific

* Calibration curve and linearity range:

Standard solutions were preparedat concentrations from 30 µg/mL to 240 µg/mL

Each concentration was analyzed 3 times Record chromatograms and peak response

The relationship between the area and concentration of NIF was studied

Table 5: Results of calibration curve (n = 3)

Regression equation: y = 83930x - 284700

The correlation coefficient R2 = 1 indicated that a linear correlation between the

concentration of NIF and the peak area was obtained in the range of concentrations

investigated This linear range was suitable for quantifying NIF in drug - excipient

compatibility testing samples

* Precision:

- Repeatability:

6 test samples were prepared according to the selected procedure and injected into

the HPLC system (prepared by tester 1) Determined the precision by calculating the

relative standard deviation between the quantifications

Table 6: Results of method precision

Under the selected chromatographic conditions, RSD of the quantitative results in

each day of analysis wss 0.53%, which was less than 2% As such, the chosen method

guarantees accuracy

- Intermediate precision:

6 test samples were prepared by another tester (tester 2) following the selected

procedure and injected them into the HPLC system Determined the intermediate

precision by calculating the relative standard deviation between the quantifications

Table 7: Results of intermediate precision

Under the selected chromatographic conditions, RSD of the quantitative results was

analyzed by each analyst was 0.24% and 0.53%, all of them were less than 2%

By both of analysts were 0.3% which was less than 3% As such, the chosen method

guarantees precision

* Accuracy:

The accuracy of the method expressed in % recovery % recovery was determined

by calculating the amounts of standard added into the placebo powder at three different

concentration levels (80%, 100%, 120%) Each level was made in triplicate (n = 3)

Table 8: Results of accuracy test

Recovery

level

Amount of the standard added (mg)

Total peak area (µV.s)

Amount of the standard recovered (mg)

%

80%

Mean = 99.2 RSD = 0.36%

100%

Mean = 98.84 RSD = 0.38%

120%

Mean = 99.68 RSD = 0.59%

The amount of standard recovery at each concentration level was in the range of

98 - 102% compared to the amount of standard added, with the RSD of % recovery at

each concentration level < 2% indicated that the proposed method was highly

accurate

* Specified range:

The accuracy results showed that at the NIF concentration of about 80% to 120%

compared to the quantitative concentration, the quantitation method achieved good accuracy and repeatability Thus, the specified range of the method was

120 - 180 µg/mL

* Determination of limit of determination and limit of quantification:

The limit of detection and limit of quantification were estimated from signal to noise

ratio Limit of detection and limit of quantification were found to be 0.0375 µg/ml and

0.105 µg/mL, respectively

CONCLUSION

A HPLC method has been developed

to quantify NIF in drug - excipient

compatibility testing samples with

chromatographic conditions such as:

HPLC system Alliance Waters 2695D;

detector 2998 dual α Absorbance; column

(RP-C18, 5 µm, 100A0, 250 x 4.6 mm)

Phenomenex LUNA; mobile phase

MeOH:H2O (65/35, v/v); flow rate:

1 mL/min; detector PDA 237 nm; injection

volume 25 µL; column temperature: room

temperature

The proposed HPLC method was

validated by evaluating the validation

characteristics such as system suitability,

specificity - selectivity, the linearity range,

repeatability, intermediate precision,

accuracy, range, limit of detection and

limit of quantitation The analytical method

had met the requirements according to

ICH regulations and Circular 32/2018 of the Vietnam Ministry of Health This method can be used to quantify NIF in

drug - excipient compatibility testing samples

REFERENCES

1 Vietnamese Ministry of Health Circular 32/2018, Regulate the registration of drugs and drug materials

2 Uday Y.A et al Estimation of nifedipine

by reverse phase high performance liquid chromatography tablet dosage form IJPLS

2011, pp.610-612

3 Sweetman S.C et al Martindale

Pharmaceutical Press 2014, pp.1447-1455

4 Klaus Florey et al Analytical profiles of

drug substances Academic Press InC 1989, Vol 18, pp.245-253

5 ICH Harmonised Tripartite Guideline

procedures: Q2 (R1) Text and Methodology 2005.