Study on determination of nifedipine in dog plasma by ultra performance liquid chromatography with tandem mass spectrometric detection

To validate the determination method of nifedipine (NIF) in dog plasma by ultra performance liquid chromatography with tandem mass spectrometric detection (UPLC-MS/MS). The evaluation of the specificity, selectivity, lower limit of quantitation, accuracy, precision, calibration curve and the linear range, recovery, carry-over, matrix effect, stability of NIF in plasma following the guidance of US-FDA.

STUDY ON DETERMINATION OF NIFEDIPINE IN DOG PLASMA

BY ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY WITH

TANDEM MASS SPECTROMETRIC DETECTION

Tran Quang Trung 1 , Nguyen Thanh Hai 2

Trinh Van Lau 3 , Nguyen Huu Duy 1 , Nguyen Thi Dao 4

SUMMARY

Objectives: To validate the determination method of nifedipine (NIF) in dog plasma by ultra

performance liquid chromatography with tandem mass spectrometric detection (UPLC-MS/MS)

Methods: The evaluation of the specificity, selectivity, lower limit of quantitation, accuracy,

precision, calibration curve and the linear range, recovery, carry-over, matrix effect, stability of

NIF in plasma following the guidance of US-FDA Results: The UPLC-MS/MS method has been evaluated and achieved the US-FDA’s regulations Conclusion: The UPLC-MS/MS method can

be used to determine the NIF in dog plasma, which will be used in bioavailability studies of the NIF dosage forms

* Keywords: Nifedipine; UPLC-MS/MS; Dog plasma

INTRODUCTION

Nifedipine (NIF), a calcium channel

blocker, is the first dihydropyridine drug to

be clinically used to treat coronary venous

insufficiency in 1969 [2] It is widely used

in the treatment of angina, hypertension

and other vascular disorders [1, 3, 4]

According to the Biopharmaceutics

Classification System (BCS), NIF is a

typical Class II compound with high

permeability, lipid solubility, which is rapidly

and completely absorbed from the gastrointestinal tract after oral administration It has a very low bioavailability of 45 - 75%, mainly due to pre-systemic metabolism, which may result in very low plasma concentrations [1] Several methods for determination

of NIF in plasma samples by gas chromatography, voltammetric method or HPLC with UV detection and electrochemical

detection have been reported [5, 6, 7, 8, 9]

1

Training Institute of Pharmacy, Vietnam Military Medical University

2

School of Medicine and Pharmacy, Vietnam National University, Hanoi

3

National Institute of Drug Quality Control

4

Thanh Phat Pharmacy Joint Stock Company

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

Date received: 29/6/2020

Date accepted: 06/7/2020

However, these methods are not

sensitive enough to determine NIF in low

plasma concentrations In order to conduct

the bioavailability studies of the NIF

dosage forms by rapid, highly sensitive

and accurate method, based on existing

equipments, especially with very low

plasma NIF concentration, we conducted

this study aiming: To determine NIF in

dog plasma by UPLC-MS/MS

MATERIALS AND METHODS

1 Materials and equipments

* Materials, solvents and chemicals:

- Standards:

Reference standard of NIF (potency

99.82%) was provided by National Institute

of Drug Quality Control Control Number:

WS 0216200.02

Internal standard of Glibenclamid

(potency 100.01%) was provided by

National Institute of Drug Quality Control

Control Number: 0103129, moisture content:

0.09%

- Solvents and chemicals:

HPLC grade acetonitrile, methanol, acid

formic were procured from Merck Ltd

Analytical grade chloroform was procured

from Merck Ltd

- Blank plasma: Dog plasma without

analytical substance were supplied by

Animal Board, Vietnam Military Medical

University Lactose monohydrate, sodium

chloride, magnesium stearate, PVP K30,

red iron oxide were sourced from China

and all meet USP 38 standards

* Equipments:

All analytical equipments meet ISO/IEC

17025 - 2005 and GLP standard, including: Quantum Ultra Triple Quadrupole Mass Spectrometer system Alliance Waters, Detector Xevo TQD (USA), electrospray ionization (ESI) source; Hypersil Gold column (C18, 1.8 µm, 50 x 2.1 mm); Analytical balance Mettler Toledo (precision 0.01 mg); Elmasonic S100H ultrasonic shaker (Germany); HS 260

horizontal shaker (IKA, Germany);

Genious 3 vortex shaker (IKA, Germany); deep fridge (Panasonic, Japan) and other common instruments of appropriate accuracy in laboratory

2 Methods

* Chromatographic conditions:

- Analytical device: UPLC-MS/MS system (Water)

- Chromatographic column: C18;

50 x 2.1 mm; 1.9 µm Column temperature:

400C

- Mobile phase: MeCN: formic acid 0.1% (90 : 10, v/v)

- Flow rate: 0.2 mL/min

- Detector: Xevo TQD

- Injection size: 5 µL

- Autosampler temperature: 200C

* Mass conditions:

+ Mass spectrometry type: MS/MS, electrospray ionization source ESI (+)

+ Parameters of mass spectrometry device for NIF and glibenclamid detection are presented in the table 1

Table 1: Parameters of mass spectrometry detector for qualitative and quantitative

NIF and internal standard glibenclamid

Analytical substance

Parameters

* Sample preparation:

Allow the plasma sample to thaw at

room temperature Take sufficiently 0.5

mL of the plasma sample, transfer it into a

15 mL glass vial and add exactly 50 µL of

internal standard solution (glibenclamide

solution of 0.4 µg/mL) It was vortexed for

5 seconds and extracted with 4 mL of

chloroform for 5 minutes in a horizontal

shaker at 300 times/minute Samples

were centrifuged at 4,000 rpm for 5

minutes Take exactly 2 mL of the organic

phase into a 15 mL glass vial and

evaporated without heat to dry under

nitrogen stream Dissolve the residue in 0.5 mL of mobile phase and inject into the UPLC-MS/MS system

* Method validation:

The proposed UPLC-MS/MS method

of NIF analysis was validated to assess fundamental parameters such as: Specificity, selectivity, lower limit of quantitation, accuracy, precision, calibration curve and the linear range, recovery, carry-over, matrix effect, stability of NIF in plasma, according to the recommendations

of the current United State Food and Drug Administration (U.S FDA) [10]

RESULTS AND DISSCUSION

1 Specificity and selectivity

min

%

0

100

MRM of 2 channels,ES+

494.20 > 369.12 DHCL-BLANK4 Smooth(Mn,1x2)

3.312e+002

Glibenclamid 0.74 39 Glibenclamid;0.74;39 0.18

0.01

1.40 1.15

1.03

min

%

0

100

MRM of 2 channels,ES+

347.07 > 315.02 DHCL-BLANK4 Smooth(Mn,1x2)

1.469e+003

nifedipin 0.74 104

0.01

nifedipin 0.74 104

Figure 1: Chromatogram of blank plasma sample

min

%

0

100

MRM of 2 channels,ES+

494.20 > 369.12 DHCL-LLOQ4 Smooth(Mn,1x2)

2.230e+005

Glibenclamid 0.75 13639

min

%

0

100

MRM of 2 channels,ES+

347.07 > 315.02 DHCL-LLOQ4 Smooth(Mn,1x2)

2.672e+004

nifedipin 0.74 1717

Figure 2: Chromatogram of spiked sample containing reference standard of NIF and

internal standard of glibenclamide at LLOQ concentration of 0.5 ng/mL and

concentration of 40 ng/mL, respectively

Analyze the blank plasma and spiked samples containing reference standard of NIF

at a concentration of about 0.5 ng/mL and internal standard of glibenclamide following the developed method

The results showed that peak area response of blank plasma sample at the retention time corresponding to that of NIF (0.74 minutes) was 7% smaller than peak area response of NIF in concentration of 0.5 ng/mL, respectively and peak area response of blank plasma sample at the retention time corresponding to that of GLI (0.75 minutes) did not exceed 1% of peak area response of GLI Hence, the procedure is specific and selective to NIF and GLI in accordance with the regulations of bioanalytical method

2 Calibration curve and the linear range

Table 2: The linearity of the method obtained after regression analysis

CC Conc

(ng/mL)

Ratio

NIF/GLI

Conc

(ng/mL)

Ratio

NIF/GLI

Conc

(ng/mL)

Ratio

NIF/GLI

Conc

(ng/mL)

Ratio

NIF/GLI

Conc

(ng/mL)

Ratio

NIF/GLI

S1 0.5 0.116 0.5 0.131 0.5 0.141 0.5 0.132 0.5 0.135 S2 1.0 0.227 1.0 0.234 1.0 0.267 1.0 0.25 1.0 0.26 S3 2.0 0.426 2.0 0.435 2.0 0.507 2.0 0.501 2.0 0.487 S4 5.0 1.020 5.0 1.056 5.0 1.225 5.0 1.161 5.0 1.178 S5 10.0 1.981 10.0 2.117 10.0 2.357 10.0 2.285 10.0 2.385 S6 20.1 4.122 20.1 4.214 20.1 4.674 20.0 4.751 20.1 4.678 S7 40.1 7.681 40.1 7.984 40.1 8.930 40.1 8.803 40.2 8.983 S8 80.2 15.343 80.2 16.259 80.2 18.005 80.2 17.786 80.3 18.144 S9 100.3 19.857 100.3 20.676 100.3 22.186 100.2 23.299 100.4 23.044

(Conc.: Concentration)

Table 3: Accuracy of standard samples

Precision compared to the theoretical concentration (% )

CC

Plasma samples containing reference

standard of NIF were prepared at

concentrations from 0.5 - 100 ng/mL

Analyze the samples following the developed

method The correlation between the

concentration of NIF (x) and the peak

area ratio of NIF and GLI (y) has been

studied by linear regression method, used

weighting coefficient (1/concentration2)

The results indicated that a linear

correlation between the concentration of

NIF and the peak area ratio of NIF and GLI was obtained in the range of

concentrations from 0.5 - 100 ng/mL with

the correlation coefficient r > 0.99 The

concentrations of NIF determined from the calibration curve compared with the theoretical concentrations were within the accepted limits in accordance with the regulations of bioanalytical method (80 - 120% for the lowest concentration,

85 - 115% for the remaining concentrations)

3 Lower limit of quantitation

Table 4: Validation results of lower quantitative limits of the method

Sample

Theoretical

concentrations

(ng/mL)

Concentrations of NIF determined from the calibration curve

(ng/mL)

Compared to the actual concentration

(%)

Signal to noise ratio (S/N)

5

0.5

Analyze the blank plasma and spiked samples containing reference standard of NIF

at an exact concentration of approximately 0.5 ng/mL and internal standard of glibenclamide (LLOQ sample) The concentration of NIF in LLOQ samples was determined from the calibration curve, which was carried out in parallel under the same conditions

The results showed that S/N value of NIF peak in LLOQ samples were greater than

10 Mean ratios of NIF concentration in the samples which were determined from the calibration curve compared with the theoretical concentrations were within the range of

80 - 120%, which indicates that the results met the requirements of the lower quantitative limit of bioanalytical method following the guidance of US-FDA

4 Accuracy and precision

Table 5: Validation results of intra-day precision and accuracy

LLOQ (0.5 ng/mL)

LQC (1.5 ng/mL)

HQC ( 50.2 ng/mL)

HQC (75.3 ng/mL) Sample

Conc

(ng/mL)

(a)

Accuracy (%) (b)

Conc

(ng/mL)

(a)

Accuracy (%) (b)

Conc

(ng/mL)

(a)

Accuracy (%) (b)

Conc

(ng/mL)

(a)

Accuracy (%) (b)

1 0.6 113.5 1.6 105.6 43.1 85.8 71.9 95.5

2 0.5 100.4 1.6 109.4 49.6 98.9 70.9 94.2

3 0.6 112.2 1.7 116.2 51.1 101.8 67.7 89.9

4 0.6 119.5 1.6 108.5 50.0 99.5 70.9 94.2

5 0.6 123.8 1.6 106.6 48.4 96.5 71.2 94.6 Mean 0.6 113.9 1.6 109.3 48.4 96.5 70.5 93.7

(Conc.: Concentration)

Table 6: Validation results of inter-day precision and accuracy

LLOQ (0.5 ng/mL)

LQC (1.5 ng/mL)

HQC ( 50.2 ng/mL)

HQC (75.3 ng/mL) Sample

Conc

(ng/mL)

(a)

Accuracy (%) (b)

Conc

(ng/mL)

(a)

Accuracy (%) (b)

Conc

(ng/mL)

(a)

Accuracy (%) (b)

Conc

(ng/mL)

(a)

Accuracy (%) (b)

0.6 113.5 1.6 105.6 43.1 85.8 71.9 95.5 0.5 100.4 1.6 109.4 49.6 98.9 70.9 94.2 0.6 112.2 1.7 116.2 51.1 101.8 67.7 89.9 0.6 119.5 1.6 108.5 50.0 99.5 70.9 94.2 Day 1

0.6 123.8 1.6 106.6 48.4 96.5 71.2 94.6 0.5 97.7 1.6 106.6 46.9 93.4 74.8 99.3 0.5 100.2 1.5 100.2 48.1 95.8 70.7 93.9 0.5 107.7 1.6 107.8 49.7 98.9 68.2 90.6 0.5 107.7 1.7 110.1 49.5 98.5 70.5 93.6 Day 2

0.6 119.1 1.5 102.4 48.0 95.6 70.3 93.4

0.5 106.5 1.5 103.0 42.4 84.5 67.3 89.4 0.6 110.4 1.5 98.2 44.7 89.1 65.2 86.6 0.5 108.9 1.4 96.4 43.1 85.8 62.4 82.8 0.4 83.4 1.4 91.5 44.2 88.1 63.7 84.7 Day 3

0.5 101.8 1.4 94.0 39.9 79.4 65.2 86.6 Mean 0.5 107.5 1.5 103.8 46.6 92.8 68.7 91.3

(Conc.: Concentration)

Intra-day or inter-day precision and accuracy were validated by five replicate analysis of LLOQ, LQC, MQC and HQC samples containing NIF at concentrations of 0.5; 1.5; 50; 75 ng/mL, respectively The content of NIF in the samples was determined from the calibration curve and the percentage ratio between the concentration determined from the calibration curve and the theoretical concentration

The results indicated that mean accuracy for intra-day and inter-day batch ranged from 93.7 - 113.9% for NIF and 91.3 - 107.5% for NIF, respectively Similarly, intra-day and inter-day precisions ranged from 2.3 - 7.8% and 5.0 - 11.6% for NIF, which indicates that the results met the requirements of the intra-day or inter-day precision and accuracy of bioanalytical method following the guidance of US-FDA (≤ 20% at LLOQ and ≤ 15% for others)

5 Recovery

Table 7: Results of recovery percentage of NIF and GLI

Recovery percentage (%)

104.3

Recovery percentage of NIF and GLI were determined by comparing the mean peak area of NIF and GLI obtained by injecting five extracted samples of LQC, MQC and HQC with the mean peak area obtained by injection of respective aqueous standard solutions in blank matrix

The results showed that sample preparation method with high and stable extraction efficiency (the difference between concentrations is not more than 10%)

6 Matrix effect

Table 8: Results of matrix effect

Sample

Collect blank plasma samples of various origins Extract the blank plasma samples according to the developed procedure for obtaining the respective sample matrix solutions Prepare the standards at concentrations of LQC and HQC in sample matrix solutions in parallel with the standards at these in mobile phase Matrix effect was evaluated through MFNIF/MFIS ratio which was determined by comparing NIF area response and GLI area response of LQC and HQC in sample matrix solutions with NIF area response and GLI area response of these in mobile phase

The results showed that there was no difference between the different sample matrix with CV < 15%

7 Carry-over

Table 9: Results of carry-over

Mean LLOQ Sample

Conclusion

1 110 13 1374 10699 0,080 0,0012 Qualified

2 165 42 1374 10536 0,119 0,0040 Qualified

3 133 40 1377 10610 0,096 0,0038 Qualified

4 153 35 1319 10526 0,111 0,0033 Qualified

5 76 5 1461 10366 0,055 0,0005 Qualified

Prepare 6 blank plasma samples, 5 LLOQ samples and 6 ULOQ samples containing reference standard of NIF at a concentration of about 0.5 ng/mL, 100 ng/mL following the developed method Inject the blank plasma samples after each ULOQ sample in UPLC-MS/MS system

The results indicated that peak area response of blank plasma sample at the retention time corresponding to that of NIF was 20% smaller than peak area response

of NIF in LLOQ concentration of 0.5 ng/mL and peak area response of blank plasma sample at the retention time corresponding to that of IS was 5% smaller than peak area response of IS in LLOQ concentration of 0.5 ng/mL, which reveals that the results met the requirements of carry-over of bioanalytical method in accordance with the guidance

of US-FDA

8 Stability of NIF in plasma

Table 10: Results of stability of NIF in plasma

Theoretical concentration (ng/mL)

Concentration after storage

(ng/mL; n = 3)

Difference (%)

Three freeze-thaw cycles

Short-term stability

(5 hour; room temparature) HQC 75.3 64.0 -15.0

Autosampler stability

Long - term stability

(-600C ± -50C, 45 days)

Stability of NIF in plasma was investigated using spiked samples at two different concentration levels (LQC, HQC) prepared in triplicate Stability of NIF in plasma was evaluated by preparing the concentration of NIF in storage samples with theoretical concetration

The results showed that concentrations of NIF in the LQC and HQC samples that were stored for three freeze-thaw cycles, five hours in room temparature, 24 hours in autosampler and 45 days in temperature of -60 ± 50C differs from the theoretical concentration by not more than 15% Therefore, NIF was stable in plasma under the above storage conditions