Determination of lansoprazole in dog plasma using reversed phase high performance liquid chromatography

Objectives: To determine lansoprazole (LPZ) in dog plasma. Methods: A single-step liquidliquid extraction with tert-butyl methyl ether was used for LPZ extraction from plasma samples obtained. LPZ was determined by using reversed phase-high performance liquid chromatography (HPLC).

DETERMINATION OF LANSOPRAZOLE IN DOG PLASMA USING

REVERSED PHASE-HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

Luong Quang Anh*; Vu Ngoc Thang** Tran Nguyen Ha***; Nguyen Ngoc Chien***

SUMMARY

Objectives: To determine lansoprazole (LPZ) in dog plasma Methods: A single-step liquid-liquid extraction with tert-butyl methyl ether was used for LPZ extraction from plasma samples obtained LPZ was determined by using reversed phase-high performance liquid chromatography (HPLC) Results: LPZ and internal standard were separated by using an isocratic mobile phase on reversed phase waters symmetry C18 column with the ultraviolet detection at 285 nm Linearity range was 0.05 - 3.0 µg/mL (r > 0.999 of the regression line) The lower limitation of quantitation was 50 ng/mL Mean percentage recoveries were from 97.67 to 101.02% HPLC method was validated with intra-day and inter-day precision of 2.27 - 5.96% and 3.79 - 4.82%, respectively; accuracy was from 97.50 to 101.68% LPZ was stable in plasma samples stored at four different degradation conditions Conclusions: The method was successfully applied to determine LPZ in dog plasma samples

* Keywords: Lansoprazole; High performance liquid chromatography; Dog plasma; Validation

INTRODUCTION

Lansoprazole, a proton pump inhibitor,

reduces gastric acidity, an important factor

in treating acid-related disorders such as

gastric ulcers, duodenal ulcers, and reflux

oesophagitis LPZ is used to treat

gastro-esophageal reflux, ulcers, acid-related

dyspepsia, etc [6] LPZ is 97% bound to

plasma proteins and shows its effectiveness

correlating to drug concentration in object

plasma To estimate LPZ in plasma, some studies revealed that HPLC method was necessary [1, 2, 3, 5] However, there have been studies for determination of LPZ in plasma in Vietnam up to now Hence, we have studied to determine LPZ

in dog plasma corresponding with drug quality control requirements in fluids This research was also conducted to contribute

to further bioequivalence studies

* National Institute of Burns

** Military Institute of Pharmaceutical Analysis and Research

*** Hanoi University of Pharmacy

Corresponding author: Luong Quang Anh (luongquanganh@vmmu.edu.vn)

Date received: 18/11/2017 Date accepted: 18/01/2018

MATERIALS AND METHODS

1 Materials and equipments

Standard LPZ was provided from

National Institute of Drug Quality Control

with its content of 99.5% Pantoprazole

(PPZ) as an internal standard was

provided from Institute of Drug Quality

Control in Hochiminh City with its content

of 93.87% Methanol (MeOH), acetonitrile

(TBME) were HPLC grade and purchased

ingredients used were of analytical grade

Dog plasma was obtained from experimental

healthy beagle dogs weighted from 10 to

12 kg following a standardized feeding

process

Chromatographic measurements were

made on the HPLC system (Shimadzu,

Japan) The centrifuge machine (Eppendorf,

Germany), vortex equipment (Heidolph,

Germany) and vacuum dried centrifuge

(miVAC, England) were used for LPZ and

PPZ extraction processes All the equipments

were validated yearly by applying the

good laboratory practice criteria

2 Methods

* Preparation of stock and standard

solutions:

Stock solutions at concentration of

1 mg/mL were prepared by dissolving a

quantity of standard LPZ or internal

standard in MeOH Working standard

solutions were diluted with MeOH when

used for assay from prepared stock solutions

* Chromatographic conditions:

HPLC separation was carried out by

using a RP18 steel column (150 x 4.6 mm;

5 µm) preceded by a steel guard column (RP18, 4 x 3 mm) The UV detector was set at 285 nm The flow-rate was 1 mL/min, injection volume was 50 µL The mobile phase was a mixture of 65:35 (v/v) 0.01 M potassium dihydrophosphate buffer solution (pH was adjusted to 8.0 with triethylamine) and ACN

* Extraction of LPZ from plasma:

50 µL internal standard solution of PPZ (40 µg/mL) and 2 mL tert butyl methyl ether were added to 500 µL of each plasma sample The solution was extracted

by vortex-mixing for 3 minutes, followed

by centrifugation at 4,500 rpm/min for

10 minutes at 200C A 1 mL aliquot of the supernatant obtained was transferred to a glass tube and evaporated until dry at 300C The residue was dissolved in the solution containing 80 µL ACN and 120 µL 0.01 M potassium dihydro phosphate buffer solution (Ph was adjusted to 8.0 with triethylamine) and mixed for 2 minutes

A 50 µL aliquot was subsequently injected into the HPLC system

* Validation method:

The method was validated by application the FDA guidelines [4] with respect to specificity, linearity, accuracy and precision, extraction productivity, sample stability and lower limit of quantitation (LLOQ)

RESULTS AND DISCUSSION

1 System suitability

The HPLC system was equilibrated with the initial mobile phase composition, followed by 6 injections of the same standard solution including PPZ at 2 µg/mL and LPZ at 1 µg/mL in mobile phase

Table 1: System suitability

Samples

(min)

S

The system suitability parameters including peaks and theoretical plates met the chromatographic requirements All parameters were satisfactory with good specificity for the stability assessment of PPZ and LPZ with good retention time (%RSD < 0.1) and S ratio (%RSD < 2)

2 LPZ extraction from plasma samples

Three methods for LPZ extraction from plasma samples were performed as follows: protein precipitation by MeOH (I); liquid-liquid extraction by a mixture of diethyl ether (DE) and dicloromethane (DCM) (70:30, v/v) (II); liquid-liquid extraction by TBME (III)

Table 2: Productivities of different LPZ extraction methods from plasma samples

Concentration of 1.0 µg/mL

(n = 6)

Concentration of 2.0 µg/L

(n = 6) Methods

(Note: P = Mean of productivity)

The results showed that all extraction methods from plasma samples had considerable productivity and stability The liquid-liquid extraction by TBME showed more precision and reiteration than the two other methods Besides, the representative chromatograms of LPZ and PPZ in extraction method using TBME was balanced and sharp without impurity peaks nearby the main peaks The chromatograms also depicted straight background line and high LPZ peak Thus, the liquid-liquid extraction

by TBME has been chosen for LPZ isolation from obtained plasma samples

3 Validation method

- Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of

components that may be expected to be present in the sample matrix For

demonstrating the specificity of the method for drug assay in serum, the plasma

samples were spiked (PPZ as an internal standard was added into blank plasma

sample and LPZ plasma sample) and the representative chromatograms were shown

on figure 1, figure 2 and figure 3

Minutes

0.00

0.01

0.02

0.03

0.04

Figure 1: Chromatogram of blank plasma sample

Minutes

0.00

0.01

0.02

0.03

0.04

Figure 2: Chromatogram of blank plasma sample added

by PPZ as an internal standard

M in u tes

0 0 0

0 0 1

0 0 2

0 0 3

0 0 4

Figure 3: Chromatogram of LPZ plasma sample added by PPZ as an internal standard

The impurities did not interfere with the drug peak on figure 2 and figure 3, meanwhile, there were no impurity peaks from minutes 3 to minutes 10 on figure 1 The representative chromatograms on figure 2 and figure 3 showed that the PPZ and LPZ peaks were considerable, sharp and separately with its retention times of 4.1 and 8.2 minutes, respectively Thus, the method is specific for PPZ and LPZ

* Linearity:

The plasma samples were extracted to isolate LPZ and PPZ using the selected method The residue was dissolved in mobile phase, then an amount of aliquot was subsequently injected into HPLC system Peak area ratios between LPZ and PPZ were calculated Calibration curves were performed on 6 different days and mean calibration curve was determined Based on calibration curve, accuracy was calculated at each point of LPZ concentration The results were shown in table 3

Table 3: Range and linearity

LPZ concentrations

Linear regression

Calculated accuracy based

(Notes: y = Peak area ratio; x = LPZ concentration (µg/mL)

Linearity was established over the concentration range of 0.05 - 3.0 µg/mL for LPZ (0.05, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 3.0 µg/mL, respectively) Peak area ratios (y) of LPZ/PPZ were plotted versus LPZ respective concentrations (x) and linear regression analysis were performed on the resultant calibration curves Coefficient correlation (R2) was found to be more than 0.999 for all the analysis Calculated accuracy was in range

of 85 - 115% (from 92.26 to 102.29%)

* Precision and repeatability:

Precision and repetability assay was conducted on three samples as LQC, MQC and HQC containing LPZ following: 50 µL internal standard solution of PPZ was added

to 500 µL of LPZ plasma samples at level of 0.2, 1.5, 2.5 µg/mL, then extracted by the chosen method At each concentration, extraction of LPZ in plasma sample process was conducted in six replications on three consecutive days LPZ concentration was determined by using calibration curve in the same analytical conditions The data obtained from precise experiments were given in table 4 and table 5

Table 4: Intra-day precision and repeatability

(µg/mL)

Precision (%)

Repeatability (%RSD)

Table 5: Inter-day precision and repeatability (3 days)

(µg/mL)

Precision (%)

Repeatability (%RSD)

In all three samples (LQC, MQC, HQC), intra-day and inter-day precisions were in range of 97.50 - 101.68% The %RSD values of repeatability for the intra-day precision study and for the inter-day study were less than 10 confirming that the method was sufficiently precise for analytical requirements in fluids

* Lower limit of quantitation (LLOQ):

LLOQ is the lowest amount analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions The LLOQ value was found to be 50 ng/mL for serum with the chromatogram depicted on figure 4 and figure 5

M inu tes

0 0 0 0

0 0 0 2

0 0 0 4

0 0 0 6

0 0 0 8

Figure 4: Chromatogram of LPZ at concentration 50 ng/mL

M in u te s

-0.0 02

0.0 00

0.0 02

0.0 04

0.0 06

0.0 08

Figure 5: Chromatogram of blank serum

* LPZ stability studies:

- Stability of LPZ in stock solution:

Table 6: Stability of LPZ in stock solution

LPZ concentration (mg/mL) Stock

Max difference (%)

Comparison of LPZ concentration in stock solution in MeOH after five days stored at

2- 80C with LPZ concentration in immediate stock solution was used At 1stday, 3rdday and 5thday, LPZ in experimental stock solution was determined using the proposed method The results showed that LPZ stock solutions were stable after storage at the experimental condition

- Stability of LPZ in serum:

Table 7: Stability of LPZ in plasma samples

Stability studies

Actual LPZ concentration Mean ± SD (µg/mL, n = 6)

LPZ concentration in degradation condition*

Mean ± SD (µg/mL, n = 6)

Difference (%)

Freeze-unfreeze process

Stability after store at

Stability after store at room

Stability in autosampler

(*: LPZ concentration was calculated from calibration curve in the same analytical condition)

Stability studies were established in plasma samples containing LPZ at

concentration of 0.2 and 2.0 µg/mL LPZ concentration of samples stored at different

conditions was calculated and compared with that of initial correlative samples The

results showed that LPZ plasma samples were stable after storage at the experimental

conditions

* Extraction productivity:

- Extraction productivity of LPZ in serum:

Table 8: Extraction productivity of LPZ in serum (n = 6)

Concentration of 0.5 µg/mL

Concentration

of 1.0 µg/mL

Concentration

of 2.0 µg/mL Parameters

(Note: MP = Mobile phase)

From the data in table 8, the selected extraction method performed high and stable

productivity of LPZ in serum (from 97.67 to 101.02%, %RSD from 1.76 to 5.02)

* Extraction productivity of PPZ as an internal standard:

Assessment of PPZ extraction productivity at its concentration of 2 µg/mL was used

Table 9: Extraction productivity of PPZ in serum (n = 6)

(mAU.s)

Peak area of PPZ in serum

(mAU.s)

Productivity (%)

Mean extraction productivity of PPZ in serum was 96.35% and %RSD was 3.02 Hence, the selected extraction method was seen as an appropriate measure for determination of LPZ and PPZ internal standard from plasma samples

* Assay of commercial product:

The validated method was applied to the determination of LPZ in plasma sample obtained from experimental healthy beagle dog used a commercial capsule containing

30 mg LPZ enteric coated pellets by oral route with single dose The chromatogram of LPZ on figure 6 showed that the method was suitable for determination of LPZ in dog plasma For this reason, it is possible to apply the method in bioequivalence studies of LPZ on experimental animal as dog

Minutes

-0.02

0.00

0.02

Figure 6: Chromatogram of LPZ and PPZ in plasma sample from beagle dog

CONCLUSION

The method for determination of LPZ

in dog plasma has been established The

advantages of the method were simple

mobile phase, suitable liquid-liquid

extraction with high productivity In

addition, the method has good precision

and repeatability for application to

determine LPZ in bioequivalence studies

on experimental animals

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