Journal of Xi’an Shiyou University, Natural Science Edition ISSN : 1673-064X

Development And Validation Using Protein Precipitation

Extraction For The Estimation of Dexlansoprazole In
Spiked Human Plasma By Liquid Chromatography
Tandem Mass Spectrometry
Rinchi Bora*, S.N. MEYYANATHAN**

* Department of Pharmaceutical Analysis, JSS College of Pharmacy (JSS Academy of Higher Education and Research, Mysuru), Udhagamandalam,
Tamil Nadu, India.

Abstract- To develop a simple, precise and accurate LC-MS/MS previously reported in bulk and human plasma. It was done for
method for the estimation of dexlansoprazole in spiked human determination of DLP, simultaneous estimation and for
plasma with cost effective protein precipitation extraction pharmacokinetic study.
technique. The validation of the proposed method was done There is no reported cost effective method for the
according to the ICH and FDA guidelines. Omeprazole was used estimation of DLP by LC-MS/MS method and hence, a sensitive
as the internal standard (IS). The method was developed in and validated bioanlytical method for the estimation of DLP as
isocratic separation mode using Zorbax SB C18 column (4.6 x 150 per FDA guidelines [13-16]was undertaken.
mm, 3µm) as a stationary phase and the mobile phase consists of II. Materials and Methods
0.5 mM Ammonium acetate buffer, adjusting the pH to 3.5 using 2.1. Materials
glacial acetic acid and acetonitrile in the ratio of 30:70% V/V with DLP used as working standard was provided as a gift
a flow of 0.5 mL/min. Detection in UV was at 284 nm and it was sample from Indian Pharmacopoeia Commission, New Delhi,
carried out by triple quadrupole mass spectrometry with India and Omeprazole (Figure 2) used as internal standard was
electrospray ionization in positive mode. Proton adducts at m/z purchased from Drugs testing laboratory, JSS College of
369.95 > 251.95 and 346.00 > 198.05 to monitor Dexlansoprazole Pharmacy, Ooty, India. Acetonitrile was analytical reagent grade
and Omeprazole. The method was found to be linear over a used for LC-MS/MS purchased from Sigma Aldrich, Ammonium
concentration range of 0.5-3000 ng/mL with a regression analysis Acetate from Rankem Fine Chemical Limited and Water of LC-
of 0.9994. The percentage recovery of the present method was MS/MS grade from Milli-Q RO system (Millipore, Bedford, USA)
found to be 99.54 ± 0.28%. The method was found to be stable. were used.
The developed LC-MS/MS method will be suitable for the 2.2. Instrumentation
analysis of dexlansoprazole and applied for routine analysis in LC system coupled with tandem quadrupole mass
different quality control and research laboratories. It will be more spectrometry (Shimadzu 8030, Tokyo Japan) equipped with
precise, accurate and cost effective while comparing to the electrospray ionization (ESI) interface, LC-20AD pump, SPD-
reported method. M20 PDA detector, CTO-20AC column oven, CBM-20 alite
Index Terms- Dexlansoprazole, Omeprazole, Human Plasma, LC- controller and SIL-20AC autosampler was used. The data were
MS/MS, Validation, FDA, Electrospray Ionization recorded using Lab solution data station software. Isocratic
separation was achieved using Zorbax SB C18 column (4.6 x 50
mm, 3µm) as a stationary phase and the mobile phase consists of
I. INTRODUCTION (0.5 mM) Ammonium acetate (pH 3.5): acetonitrile (30:70 V/V)
Dexlansoprazole [DLP] {(R) - (+) – 2 - ([3- methyl – 4 - (2, 2, 2 with a flow of 0.5 mL/min and injection volume of 10 µl was
trifluoroethoxy) pyridine – 2 – yl] methylsulfinyl) - 1H - benzo [d] employed.
imidazole)} is a proton pump inhibitor (Figure 1) of the 2.3. Selection of mass range
antisecretory, substituted benzimidazole class. It is having a A 1000 ng/mL of DLP and OMP was infused into the
molecular mass of 369.69 g/mol. It is an enantiomer of mass spectrometer directly and the conditions for operation were
lansoprazole [1,2,9,10]. It is available commercially as delayed- optimized. Obtained transitions were 369.95→251.95 (Figure 5)
release capsules (30 and 60 mg). It suppresses the final stage of and 346.00→198.05 m/z (Figure 6) were used to monitor DLP
acid secretion by specifically blocking the (H+/K+)- ATPase and OMP (IS) .
present in the gastric parietal cell. It is used to treat the healing of 2.4. Preparation of solution
erosive esophagitis, in maintaining of healed erosive esophagitis About 0.77 g of ammonium acetate was weighed and
and non-erosive gastroesophageal reflux disease [3-4] (GERD) dissolved in 200 ml Milli-Q RO water to get 0.5 mM. The pH was
associated with heartburn. In January 30, 2009, it was accepted by adjusted to 3.5 with glacial acetic acid.
FDA. 2.5. Preparation of standard solutions
Literature survey reveals that Dexlansoprazole was DLP and OMP solution was prepared by dissolving
estimated by HPLC [5-8] and tandem mass spectroscopy has been accurately about 10 mg in methanol and making up the volume to
10 mL with acetonitrile and this solution was refrigerated at 2-8

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Journal of Xi’an Shiyou University, Natural Science Edition ISSN : 1673-064X

°C. Spiking of DLP solution was done and dilutions were made to In quantitative analysis by mass spectrometry with
obtain working standards (Figure 5,6), calibration curve and electrospray ionization (ESI-MS), a major limitation is matrix
quality control samples and were stored in refrigerator. effects in which the coextracted matrices with the analytes can
2.6. Preparation of sample solution and extraction cause change in the signal response, causing either enhancement
Extraction of DLP from plasma was done by protein or suppression of the in teased signal. Moreover it also may
precipitation extraction technique. Plasma is stored in containers influence on the quality of results in terms of recovery.
hermetically sealed at -60 °C till analysis. A 500 µl of aliquot was
III. Results and discussion
taken in eppendorf for analysis which was mixed with 100 µl OMP
as IS with the help of a vortex mixer. From the above solution 20 3.1. Specificity
µl was taken and DLP is extracted by using acetonitrile as To determine that the excipients used are not interfering
optimized precipitating solvent (acetonitrile). The sample was with the main compound peak, test for specificity needs to be
vortexed for a min and then centrifuged for 20 min at 7000 rpm at done. No peaks were eluted along with the retention time of DLP
room temperature. Supernatant was transferred for LC-MS/MS in addition to that it was very well resolute by compound. Hence,
analysis. (Figure 3,4) the developed method results showed that it was selective for
2.7. Method Validation determination of DLP in the formulation.
Validation of the method for specificity, linearity,
accuracy, precision, range, quantitation limit, and detection limit, 3.2. Linearity
robustness and system suitability as per the ICH and FDA The evaluation of the method to be linear was by six
guidelines [11-16]. determinations at ten concentration levels with a range of 0.5-3000
ng/mL for DLP. A calibration curve was found to be linear with a
2.7.1. Specificity
mean regression of equation (y = 0.0035x + 0.1196, r2 = 0.9994,
The analyte response measurement in the presence of other S.D.=1.28) respectively, where the response factor is the Y and the
drugs, excipients and their potential impurities to demonstrate the analyte concentration in ng/mL was the X (Figure 11).
specificity. 3.3. Accuracy
The accuracy of the method was carried for three quality
2.7.2. Linearity
control (LLOQ, LQC, MQC and HQC) samples by standard
The average of six determinations at ten concentration addition method, and the accuracy was found to be 99 ± 0.12 %.
levels covering the range of 0.5-3000 ng/mL for DLP, the Application of the developed method for the estimation of DLP
evaluation of linearity was performed. Calculation of the (Table 1).
coefficient correlation, slope and intercept values was done by 3.4. Precision
using calibration curve for linearity evaluation. The same have Calculation of the method for precision was carried by
been designed. the intra-day and inter-day precision studies at three different
concentrations and they were found to be within the limits (Table
2.7.3. Accuracy 1).
Determination of accuracy was demonstrated for 3.5. Limit of detection and Limit of quantification
standard addition method from recovery studies according to ICH The lowest limit detected for the method for DLP was at
guidelines. The pre-analyzed samples were spiked with standard 0.1 ng/mL based on the signal-to-noise ratio 3:1. Due to the
drug DLP. increase in the sensitivity of the method, quantification was done
at 0.5 ng/mL for DLP. This method found to have a high
2.7.4. Precision percentage recovery at low concentration at the acceptable limit
Evaluation of precision was carried out by inter-day and (Table 1) (Figure 9 and 10).
intra-day comparision study samples consisted of three levels 3.6. Robustness
concentration (six replicates) of LLOQ, low (LQC), medium When alteration in the condition of experiment was done,
(MQC) and high (HQC) quality controls, i.e. 0.3, 1.5, 750, 2000 no notable changes in the parameters of chromatograph were
ng/mL, respectively. Report used for precision was from the observed, proving that the developed method was found to be
regressed concentration of the percent relative standard deviation highly robust.
(%RSD). 3.7. Stability studies
Exposing to various stress condition the evaluation of
2.7.5. Limit of detection (LOD) and limit of quantification (LOQ) stability was performed. According to international stability
Determination of LOD and LOQ was assessed by the guidelines the test performed are bench top stability, freeze thaw
signal-to-noise ratio. LOD ratio was 3:1 whereas LOQ of the drug stability and long term storage stability for spiked plasma samples,
could be quantified with minimum peak area in the ratio of 10:1. autosampler stability for the processed sample and stock solution
stability for the stock solution. 1 mg/mL DLP stock solution was
2.7.6. Robustness refrigerated for 7 days and kept for 8 hours in room temperature.
The alteration in the condition of the experiment like Refrigeration of DLP in plasma for 20 days at -20°C and for 18
operators, the source of reagents, similar type column and hours in room temperature. Bench top stability was achieved by
optimized conditions like pH, mobile phase ratio and flow rate keeping the samples for 8 hours in room temperature. Three cycles
were used as assessment tool for the robustness monitoring. of freeze and thaw stability was performed. DLP proc.essed
2.7.7. Matrix effect

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Journal of Xi’an Shiyou University, Natural Science Edition ISSN : 1673-064X

sample stability was done for 60 hours in autosampler in room generation proton pump inhibitor” US National Library
temperature of Medicine National Institutes of Health, Prz
3.8. Recovery and Matrix effect Gastroenterol 4 (2015) 191–196.
The mean recovery of DLP from the spiked human [5] N. Aslam and R.A. Wright, Dexlansoprazole MR, Expert
plasma was 99.95, 100.02 and 97.01 respectively. The obtained Opin. Pharmacother, 10 (2009) 2329–2336.
value evidenced that, the no matrix influenced was observed in [6] K. K. Hotha, D.V. Bharathi, J. Banda, V.
plasma sample analysed. Ion suppression is indicated in the Venkateswarulu, Development and validation of a highly
values. Therefore, the present method is reliable. The protein sensitive LC-MS/MS method for quantitation of
precipitation method has capacity to remove the maximum plasma dexlansoprazole in human plasma: application to a
bounded. human pharmacokinetic study, Biomed. Chromatogr 2
(2012) 192-198.
IV. Conclusion [7] J. Sriharsha, S.M. Murthy, B. D.Kumar, K.Sravan, P.
Shivakumar, A. Shirisha, K . Pranusha, Method for
A novel simple, precise, accurate and a validated, liquid Development and Validation for Simultaneous
chromatography-tandem mass spectroscopy method have been Estimation of Dexlansoprazole and Meloxicam by RP-
developed and validated. The developed method can be HPLC, Pharm. Anal. Acta 5 (2015) 2153-2435.
successfully applied for the estimation of DLP in the human [8] R. Mohan, P. P. Srikumar, G.V., Rushyendra, Y.
plasma. Geetharam, Stability indicating validated novel RP-
CONFLICT OF INTEREST STATEMENT HPLC method for the estimation of dexlansoprazole in
The authors declare that there are no conflicts of interest. bulk and extended release capsules, Indo Am. J. Pharm.
ACKNOWLEDGMENT Res 3 (2013) 8457-8466.
The authors thank the Indian Pharmacopoeia [9] www.dexilant.com. Accessed on Sep 2017.
Commission, New Delhi for providing the standard [10] www.drugs.com accessed on Sep 2017
Dexlansoprazole as a gift sample. [11] ICH, Q3B validation of analytical procedures:
methodology, International Conference on
REFERENCES Harmonization, November 1996 (Accessed on Nov 2017).
[12] G. Hendriks, Review Theoretical models in LC based
[1] U.S. Department of Health and Human Services Food bioanalytical method development, J Pharm Biomed Anal
and Drug administration Centre for Drug Evaluation and 49 (2009) 1-10.
Research (CDER) Centre for Veterinary Medicine [13] International Conference on Harmonization (ICH),
(CVM), Guidance for Industry Bioanalytical Method Validation of analytical methods definitions and
Validation, May 2001 BP. terminology, ICH Q2 A, 1994.
[2] H. Nagaya, H. Satoh, Y Maki, Possible mechanism for [14] International Conference on Harmonization (ICH),
the inhibition of acid formation by proton pump Validation of analalytical methods: methodology, ICH Q2
inhibitor AG-1749 in isolated canine parietal cells, J B, 1996.
Pharmacol Exp Ther. 252 (1990) 1289-1295. [15] Guidance for Industry, Bioanalytical Method Validation,
[3] D. C. Metz, M. Vakily, T. Dixit, Dual delayed release U.S. Department of Health and Human Services Food and
formulation of dexlansoprazole MR, a novel approach to Drug Administration Center for Drug Evaluation and
overcome the limitations of conventional single release Research (CDER) Center for Veterinary Medicine (CVM)
proton pump inhibitor therapy, Aliment Pharmacol Ther. (2013).
9 (2009) 928-937. [16] http//:www/fda.gov/cder/guidance/index.htm
[4] Barbara and Piotr Radwan, Dexlansoprazole – a new-

Professor, Department of Pharmaceutical Analysis, JSS College

of Pharmacy, Ooty, Tamil nadu-643001, India.
AUTHORS Email: snmeyyanathan@jssuni.edu.in
First Author –Rinchi Bora, Research Scholar, Department of Correspondence Author – Rinchi Bora, Research Scholar,
Pharmaceutical Analysis, JSS College of Pharmacy, Ooty, Tamil Department of Pharmaceutical Analysis, JSS College of
nadu-643001, India. Pharmacy, Ooty, Tamil nadu-643001, India.
Email: himrin05@gmail.com Email: himrin05@gmail.com
ORCID: 0000-0002-0140-4125 ORCID: 0000-0002-0140-4125
Second Author – Dr. S.N. Meyyanathan, Ph.D

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Journal of Xi’an Shiyou University, Natural Science Edition ISSN : 1673-064X

Figure 1. Dexlansoprazole Figure 2. Omeprazole

Table 1: Accuracy and precision for the determination of dexlansoprazole.

QC Mean Intraday Inter-day

samples Conc.
(ng/mL) found Accuracy Precision Accuracy Precision
(ng/mL) (% N) (% CV) (%N) (% CV)

15 12.83 96.00 2.83 84.0 2.23

750 720.7 96.81 2.78 96.36 2.92

2000 1994 98.83 0.96 98.14 2.03

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1:Omeprazole (IS) 346.00>198.05(+) CE: -12.0

225

200

175

150

125

100

75

50

25

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min

Figure 3. Typical LC-MS/MS separation Blank plasma and IS spectra

2:Dexlansoprozole 369.95>251.95(+) CE: -12.0

400

375

350

325

300

275

250

225

200

175

150

125

100

75

50

25

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min

Figure 4. Typical LC-MS/MS separation Blank plasma and DLP spectra

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Journal of Xi’an Shiyou University, Natural Science Edition ISSN : 1673-064X

Inten. (x1,000,000)
2.5
370.05

2.0

1.5

1.0
114.70 350.05
0.5 322.10
146.00 272.05 297.00 372.05 398.20
0.0
100 150 200 250 300 350 400 450 m/z
Figure 5. Typical mass spectra of DLP

Inten. (x10,000,000)
100.05
2.00 346.10

1.75

1.50

1.25

1.00
328.05
0.75

0.50
298.10
0.25 143.10 198.00 360.10
212.05 242.20 282.00
409.10 463.20
0.00
100 150 200 250 300 350 400 450 m/z
Figure 6. Typical mass spectra of OMP

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1:Omeprazole (IS) 346.00>198.05(+) CE: -12.0

1.291
6500000

6000000

5500000

5000000 A
4500000

4000000

3500000

3000000

2500000

2000000

1500000

1000000

500000

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min

175000 2:Dexlansoprozole 369.95>251.95(+) CE: -12.0 1.263

150000

B
125000

100000

75000

50000

25000

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min

(B)
Figure 7. Typical LC-MS/MS Standard chromatogram – (A) OMP (B) DLP

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Inten. (x10,000)
1.0 198.05
251.95
0.9

0.8

0.7

0.6

0.5
136.05
0.4

0.3 119.10 136.05 151.10

0.2

0.1

0.0
125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z
Figure 8. Typical LC-MS/MS spectra of PI scanning

1:Omeprazole (IS) 346.00>198.05(+) CE: -12.0

1.260
3500000

3250000

3000000

2750000

2500000

2250000

2000000

1750000

1500000

1250000

1000000

750000

500000

250000

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min

100000 2:Dexlansoprozole 369.95>251.95(+) CE: -12.0

1.293

90000

80000

70000

60000

50000

40000

30000

20000

10000

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min

Figure 9. Typical LC-MS/MS spectra for LOQ samples

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1:Omeprazole (IS) 346.00>198.05(+) CE: -12.0

1.283
10000

9000

8000

7000

6000

5000

4000

3000

2000

1000

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min

2:Dexlansoprozole 369.95>251.95(+) CE: -12.0

1.262
22500

20000

17500

15000

12500

10000

7500

5000

2500

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min

Figure 10. Typical LC-MS/MS spectra for LOD samples

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12
y = 0.0035x + 0.1196
10
R² = 0.9994
8
Response Factor

0
0 500 1000 1500 2000 2500 3000 3500

Concentration (ng/mL)

Figure 11. Linearity curve of DLP in plasma

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