Received: 26 January 2018
Revised: 9 April 2018
Accepted: 18 April 2018
DOI: 10.1002/bmc.4270
RESEARCH ARTICLE
LC–MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine in dried blood spots Pavan Kumar Prathipati Pharmacy Practice Department, Creighton University School of Pharmacy and Health Professions, Omaha, Nebraska, USA Correspondence Pavan Kumar Prathipati, Pharmacy Practice Department, Creighton University School of Pharmacy and Health Professions, Omaha, Nebraska, USA. Email:
[email protected]
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Subhra Mandal
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Christopher J. Destache
Abstract A simple, short, and rugged LC–MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine was developed and validated. Dried blood spots were prepared with 25 μL of spiked whole blood. A 3 mm punch was extracted with methanol containing labeled internal standards. Ten microliters was injected into the LC–MS/MS using isocratic mobile phase composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (45: 55 v/v) at a flow rate
Funding information National Institute of Allergy and Infectious Diseases, Grant/Award Number: RO1 AI117740‐01; National Center for Research Resources, Grant/Award Number: G20RR024001; NIH, Grant/Award Number: RO1 AI117740‐01
of 0.25 mL/min. The method was validated in the range of 10–2000 ng/mL for all four analytes. The intra‐assay accuracy (RE) of the method was −4.73–4.78, 1.35–2.89, −8.89 to −0.49 and − 1.40–1.81 for tenofovir, emtricitabine, elvitegravir and rilpivirine, respectively. The inter‐assay accuracy was within ±15% of nominal and precision (CV) was 0.9988
>0.9985
TFV, Tenofovir; FTC, emtricitabine; EVG, elvitegravir; RPV, rilpivirine.
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FIGURE 2 Hematocrit effect on TFV, FTC, EVG and RPV (black dots represent the measured concentration of LQC and HQC at 35, 50 and 70% hematocrit values) TABLE 3
Hematocrit effect analysis Hematocrit (%)
Hematocrit (%)
Analyte
Parameter
35 50 LQC (10 ng/mL)
TFV
Mean concentration (ng/mL; n = 3) Accuracy (RE, %) Precision (CV, %)
28.94 −3.53 3.65
30.96 3.20 2.14
31.92 6.40 0.98
1596.60 −11.30 2.80
1663.20 −7.60 3.37
1772.40 −1.53 1.62
FTC
Mean concentration (ng/mL; n = 3) Accuracy (RE, %) Precision (CV, %)
28.67 −4.43 5.65
31.85 6.17 0.65
33.11 10.37 1.10
1613.40 −10.37 0.79
1743.60 −3.13 0.34
1917.60 6.53 1.39
EVG
Mean concentration (ng/mL; n = 3) Accuracy (RE, %) Precision (CV, %)
27.48 −8.40 1.29
28.87 −3.77 3.43
32.21 7.37 1.94
1557.00 −13.50 0.85
1609.80 −10.57 0.61
1648.20 −8.43 1.41
RPV
Mean concentration (ng/mL; n = 3) Accuracy (RE, %) Precision (CV, %)
28.89 −3.71 1.91
31.90 6.32 1.20
33.43 11.42 3.40
1578.00 −12.33 1.42
1705.68 −5.24 1.23
1908.00 6.00 2.30
FIGURE 3
70
35 50 HQC (1800 ng/mL)
70
Correlation of dried blood spot (DBS) vs plasma concentration of FTC (a) and EVG (b)
methods have a longer run time (≥6 mins) than our method (4 min).
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CO NC LUSIO N
Zheng et al. used isocratic mobile phase with high aqueous content (99.5% v/v) and Waitt et al. used a gradient program starting at 95%
The developed method for simultaneous quantification of TFV, FTC,
v/v aqueous phase, whereas we used isocratic mobile phase with
EVG, and RPV in DBS was demonstrated to be accurate, precise and
45% aqueous, which is highly compatible for mass spectrometer. We
rugged. The DBS specific validation parameters like hematocrit, spot
have quantified elvitegravir and rilpivirine, which is unique compared
volume and punch location have demonstrated that this method can
with reported methods.
be used as an alternative to plasma. The correlation between DBS
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and plasma was demonstrated for emtricitabine and elvitegravir in unknown samples. This method can be conveniently applied to current human trials on a combination of these drugs, especially in low‐income countries. ACKNOWLEDGMENTS This work was supported by NIH grant RO1 AI117740‐01 to C.J.D. The Animal Research Facility is supported by grant number G20RR024001 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. ORCID Pavan Kumar Prathipati
http://orcid.org/0000-0002-2463-3350
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How to cite this article: Prathipati PK, Mandal S, Destache CJ. LC–MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine in dried blood spots. Biomedical Chromatography. 2018;e4270. https:// doi.org/10.1002/bmc.4270
