MS with application in a maternal-fetal pharmacokinetic study

Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba

Determination of raltegravir and raltegravir glucuronide in human plasma and urine by LC–MS/MS with application in a maternal-fetal pharmacokinetic study Fernanda de Lima Moreira a , Maria Paula Marques a , Geraldo Duarte b , Vera Lucia Lanchote a,∗ a Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil b Departamento de Obstetrícia e Ginecologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

a r t i c l e

i n f o

Article history: Received 5 July 2019 Received in revised form 22 August 2019 Accepted 26 August 2019 Available online 27 August 2019 Keywords: Raltegravir Raltegravir glucuronide LC–MS/MS Pregnancy

a b s t r a c t Raltegravir (RAL) is a HIV-integrase inhibitor recommended for treatment of HIV type 1 infection during pregnancy. The elimination of RAL to RAL glucuronide (RAL GLU) is mediated primarily by UDP glucuronosyltransferase 1A1 (UGT1A1). The present study shows the development and validation of 4 different methods for the analysis of RAL and RAL GLU in plasma and in urine samples. The methods were applied to evaluate the maternal-fetal pharmacokinetics of RAL and RAL GLU in a HIV-infected pregnant woman receiving RAL 400 mg twice daily. The sample preparation for RAL and RAL GLU analysis in 25 ␮L plasma and 100 ␮L diluted urine (10-fold with water containing 0.1% formic acid) were carried out by protein precipitation procedure. RAL and RAL GLU generate similar product mass fragments and require separation in the chromatographic system, so a suitable resolution was achieved for unchanged RAL and RAL GLU employing Ascentis Express C18 (75 × 4.6 mm, 2.7 ␮m) for both plasma and urine samples. The methods showed linearities at the ranges of 0.1–13.5 ␮g/mL RAL and 0.15–19.5 ␮g/mL RAL GLU in urine and 10–2000 ng/mL RAL and 2.5–800 RAL GLU in plasma. Precise and accurate evaluation showed coefficients of variation and relative errors ≤ 15%. The methods have been successfully applied in a maternal-fetal pharmacokinetic study. © 2019 Published by Elsevier B.V.

1. Introduction Raltegravir (RAL) is the first FDA approved HIV-integrase inhibitor for treatment of HIV type 1 infection during pregnancy in Brazil, US and European. RAL is recommended to be administered in combination with 2 nucleoside/nucleotide reverse transcriptase inhibitors [1–3]. RAL is considered to be safe during pregnancy [4,5] with a low incidence of adverse effects and considered to be effective showing viral load < 400 copies/mL during delivery [4,6]. The elimination of RAL is mediated primarily by UDP glucuronosyltransferase 1A1 (UGT1A1), producing the RAL glucuronide (RAL GLU) metabolite [7]. It is reported that 32% of the RAL dose is

∗ Corresponding author at: Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Avenida do Café s.n. Campus da USP, 14040-903, Ribeirão Preto, SP, Brazil. E-mail address: [email protected] (V.L. Lanchote). https://doi.org/10.1016/j.jpba.2019.112838 0731-7085/© 2019 Published by Elsevier B.V.

excreted in the urine, being 9% as unchanged drug, and 23% as the metabolite RAL GLU [7]. Considering that UGT1A1 activity and expression is associated with RAL exposure, the UGT1A1*28 carriers show higher plasma concentrations of RAL when compared to wild-type genotype [8]. Furthermore, RAL plasma concentrations are increased in patients with advanced liver cirrhosis (Child–Pugh C), probably due the lower UGT1A1 activity when compared with healthy volunteers [9]. There is a significant inter- and intra-patient variability in the pharmacokinetic parameters of RAL in both non-pregnant population and pregnant women [5,10–14]. RAL placenta transfer is effective with fetal-maternal ratio ranged from 1 to 3.48 [4,5,13]. There is no data about RAL GLU concentrations in HIV-infected pregnant women, as well as, transplacental transfer data of this metabolite. The determination of RAL GLU concentrations should give information about the role of UGT1A1 activity during pregnancy.

2

F.d.L. Moreira, M.P. Marques, G. Duarte et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

There are some validated methods for simultaneous determination of RAL with other antiretrovirals in human plasma using LC–MS/MS [15–22]. Nevertheless, few papers have described the quantification of RAL GLU in human plasma [23,24]. The chromatographic separation of RAL and RAL GLU is necessary in order to avoid the overestimation of RAL concentration, since there is an in-source transformation phenomenon that causes interference of RAL GLU in RAL analysis [15,23,25,26]. Despite of the numerous papers published about the determination of RAL in human plasma, there is not validated methods published for analysis of RAL and/or RAL GLU in urine using LC–MS/MS. The present study shows the development, validation and clinical application of the methods for the analysis of RAL and RAL GLU in plasma and urine using LC–MS/MS instruments. The methods were applied to evaluate the maternal-fetal pharmacokinetics of RAL and RAL GLU in a HIV-infected pregnant woman receiving RAL 400 mg twice daily.

2. Materials and methods 2.1. Chemicals Raltegravir, raltegravir glucuronide, raltegravir D3 (RAL D3), raltegravir D3 glucuronide (RAL D3 GLU) were purchased from Toronto Research Chemicals, Inc (Toronto, ON, Canada). Methanol (HPLC grade), acetonitrile (HPLC grade) and formic acid (analytical grade) were obtained from J. T. Baker (Philipsburg, NJ, USA). Ultrapure water was obtained from a Milli-Q® Sinergy UV water purification system (Millipore, Burlington, MA, USA).

2.2. Preparation of stock and standard working solutions All the stock and working solutions of RAL, RAL D3, RAL GLU and RAL D3 GLU were diluted in methanol as described in Table 1 and stored at −20 ◦ C. 2.3. Urine sample preparation The sample preparation for RAL and RAL GLU analysis in urine were carried out by protein precipitation procedure. Briefly, 100 ␮L diluted urine (10-fold with water containing 0.1% formic acid) was transferred into a 1.5-mL Eppendorf tube. After addition of 25 ␮L IS, methanol (75 ␮L for RAL analysis and 25 ␮L for RAL GLU analysis) and 150 ␮L acetonitrile, the tubes were vortexed for 10 s and finally centrifuged at +4 ◦ C for 10 min at 21,500 x g (15,000 rpm) on a Hitachi CT15RE centrifuge (Tokyo Japan). A 100 ␮L aliquot of the supernatant was collected and transferred into 250 ␮L glass inserts containing 100 ␮L water with 0.1% formic acid. A volume of 25 ␮L was injected into LC–MS/MS system.

2.4. Plasma sample preparation The sample preparation for RAL and RAL GLU analysis in patient plasma samples were carried out by protein precipitation procedure. Briefly, 25 ␮L of patient plasma samples was transferred into a 1.5-mL Eppendorf tube. After addition of 25 ␮L IS and 25 ␮L methanol and 250 ␮L acetonitrile, the tubes were vortexed for 10 s and finally centrifuged at +4 ◦ C for 15 min at 21,500 x g (15,000 rpm) on a Hitachi CT15RE centrifuge (Tokyo Japan). A 100 ␮L aliquot of the supernatant was collected and transferred into 250 ␮L glass inserts containing 100 ␮L water. For RAL analysis, 2 ␮L was injected, while for RAL GLU analysis, 10 ␮L was injected into LC–MS/MS system.

2.5. LC–MS/MS instrumentation for urine analysis The LC–MS/MS system for urine analysis consisted of a quaternary pump equipped with Waters e2695 separation module auto-sample injector (Waters Corp, Milford, MA USA) and column oven CTOASVP (Shimadzu Corp, Kyoto, Japan) and Quattro Micro API triple quadrupole equipped with ESI (Waters Corp, Milford, MA, USA). The RAL and RAL GLU were analyzed using the same chromatographic conditions, employing Ascentis Express C18 (75 x 4.6 mm, 2.7 ␮m) set at 24 ◦ C with flow rate of 0.2 mL/min and isocratic elution with the mobile phase consisting of acetonitrile containing 0.1% formic acid: water containing 0.1% formic acid (60:40, v/v). The mass spectrometer system for analysis of RAL in urine was operated in the positive ion mode. The capillary voltage of electrospray interface was 3.0 kV. The source and the desolvation temperatures were kept at 120 and 400 ◦ C, respectively. The collision gas (argon) was employed at a pressure of approximately 3.35 × 10−3 mbar. The voltage of the cone was kept at 30 V and collision energy of 30 eV for RAL and RAL D3 (IS). The protonated ions [M+H]+ and their respective ion products were monitored in the 445.5 > 109 m/z transition for RAL and 448.5 > 109 m/z for RAL D3. The mass spectrometer system for analysis of RAL GLU in urine was operated in the positive ion mode. The capillary voltage of electrospray interface was 3.0 kV. The source and the desolvation temperatures were kept at 120 and 200 ◦ C, respectively. The collision gas (argon) was employed at a pressure of approximately 3.0 × 10−3 mbar. The voltage of the cone was kept at 30 V and collision energy of 20 eV for RAL GLU and RAL D3 GLU (IS). The protonated ions [M + Na]+ and their respective ion products were monitored in the 643.0 > 467.0 m/z transition for RAL GLU and 646.0 > 470 m/z for RAL D3 GLU. 2.6. LC–MS/MS instrumentation for plasma analysis The LC–MS/MS system for plasma analysis consisted of the Acquity UPLC® H-Class chromatographic system coupled to a Xevo TQ-S® triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with Zspray® electrospray ionization (ESI). The MassLynx version 4.1 software (Waters, Milford, USA) was used for data acquisition and sample quantitation. The analytes were separated with the Ascentis Express C18 (75 x 4.6 mm, 2.7 ␮m) set at 24 ◦ C and with flow rate of 0.2 mL/min. RAL was determined using isocratic elution with the mobile phase consisting of acetonitrile containing 0.1% formic acid: water containing 0.1% formic acid (60:40, v/v). The run time was 7 min. The initial 4 min eluate was directed to the waste and then switched to mass spectrometry. The mass spectrometer system was operated in the positive ion mode. The capillary voltage of electrospray interface was 3.5 kV. The source and desolvation temperatures were kept at 150 and 250 ◦ C, respectively. The nebulization gas (nitrogen) was used at the flow rate of 1000 L/h. The collision gas (argon) was employed at a pressure of approximately 7 × 10−4 mbar. The voltage of the cone was kept at 20 V and collision energy of 30 eV for RAL and RAL D3 (IS). The protonated ions [M+H]+ and their respective ion products were monitored in the 445.5 > 109 m/z transition for RAL and 448.5 > 109 m/z for RAL D3. The separation of RAL GLU was achieved using gradient elution. Mobile-phase solvent A consisted of acetonitrile with 0.1% formic acid and solvent B consisted of water with 0.1% formic acid. The initial mobile-phase composition of 30% solvent A was maintained for 0.1 min and increased linearly to 90% from 0.1 to 9 min. Next, solvent A was reversed to 30% within 1 min and a 2 min reequilibration step was employed. The run time was 12 min. The initial 4 min eluate was directed to the waste and then switched

F.d.L. Moreira, M.P. Marques, G. Duarte et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

3

Table 1 Concentration of working solutions and preparation of calibration curves, LLOQ and QC samples. Biological matrix

plasma

urine

analyte

Deuterated IS

working solution concentration (in methanol)

calibration range (final concentration in biological matrix)

LLOQ

Low, Medium and High QCs controls

Dilution QC* ; dilution factor

RAL

RAL D3 (40 ng/mL)

10 - 2000 ng/mL

10 - 2000 ng/mL

10 ng/mL

4000 ng/mL; 1:10

RAL GLU

RAL D3 GLU (40 ng/mL) RAL D3 (4 ␮g/mL)

2.5 - 800 ng/mL

2.5 - 800 ng/mL

2.5 ng/mL

20, 1000, 1600 ng/mL 5, 400, 600 ng/mL

2000 ng/mL; 1:10

0.4 - 54 ␮g/mL

0.1 - 13.5 ␮g/mL

0.1 ␮g/mL

RAL D3 GLU (4 ␮g/mL)

0.6 - 78 ␮g/mL

0.15 - 19.5 ␮g/mL

0.15 ␮g/mL

0.2, 6.75, 10.1 ␮g/mL 0.3, 9.75, 14.6 ␮g/mL

67.5 ␮g/mL; 1:6 130 ␮g/mL; 1:40

RAL RAL GLU

IS internal standard; RAL raltegravir; RAL GLU raltegravir glucuronide. * Dilution QC: A concentration above the ULOQ of the calibration curve was used for achieve the dilution QC.

Fig. 1. Representative chromatograms of raltegravir (A) and raltegravir D3 (IS) (4 ␮g/mL in methanol) (B): blank urine sample (1), LLOQ 0.105 ␮g/mL (2) and patient urine sample taken at 6 h after raltegravir administration (3). Retention time of raltegravir and raltegravir D3 is 4.96 min and raltegravir glucuronide is 4.23 min.

to mass spectrometry and the last 5 min eluate was directed to the waste. The mass spectrometer system was operated in the negative ion mode. The capillary voltage of electrospray interface was 3.0 kV. The source and the desolvation temperatures were kept at 150 and 450 ◦ C, respectively. The nebulization gas (nitrogen) was used at the flow rate of 1000 L/h. The collision gas (argon) was employed at a pressure of approximately 7 × 10−4 mbar. The voltage of the cone was kept at 24 V and collision energy of 30 eV for RAL GLU and RAL

D3 GLU (IS). The deprotonated ions [M−H]- and their respective ion products were monitored in the 619.0 > 316.0 m/z transition for RAL GLU and 622.0 > 319.0 m/z for RAL D3 GLU. 2.7. Analytical methods validation The validation of the methods in plasma and urine were processed according to the guideline of EMA for bioanalytical methods.

4

F.d.L. Moreira, M.P. Marques, G. Duarte et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

Fig. 2. Representative chromatograms of raltegravir glucuronide (A) and raltegravir D3 glucuronide (IS) (4 ␮g/mL in methanol) (B): blank urine sample (1), LLOQ 0.15 ␮g/mL (2) and patient urine sample taken at 6 h after raltegravir administration (3). Retention time of raltegravir glucuronide and raltegravir D3 glucuronide is 3.90 min.

Calibration standards were prepared by spiking the working solutions according to their respective biological matrix (Table 1) into blank plasma or blank urine to get the following final concentrations: RAL 10–2000 ng/mL plasma, RAL GLU 2.5–800 ng/mL plasma, RAL 0.1–13.5 ␮g/mL urine and RAL GLU 0.15–19.5 ␮g/mL urine. The calibration standards employed in urine analysis were 10-fold diluted with water containing 0.1% formic acid. Analyte to IS peak area ratios were analyzed using linear regression with a weighting factor of 1/x2 . The precision and accuracy of the method were determined with intra- and inter-assay studies employing LLOQ, low, medium, high and dilution QCs. A concentration above the ULOQ of each calibration curve was used for achieve the dilution QC. Five aliquots of each QC of RAL or RAL GLU were analyzed in the same analytical run and in three days. The precision was calculated as the coefficient of variation (CV%) within a single run (intra-assay) and between different assays (inter-assay). The acceptance criterion was within < 15% of the CV, except at LLOQ for which < 20% of the CV was acceptable. The accuracy was calculated as the percentage of deviation between nominal and obtained concentrations (% relative error - RE). The acceptance criterion

was within ± 15%, except at LLOQ for which ± 20% was acceptable. Selectivity was evaluated using aliquots of blank plasma or urine obtained from six different volunteers. The chromatograms obtained were compared to LLOQ samples. Carry-over was analyzed by injecting blank samples before and after a sample at the ULOQ. The chromatogram of the blank sample following the ULOQ standard was compared to that of the LLOQ. The matrix effect was investigated by calculating the peak area ratios of the analyte added into blank plasma extracts after the extraction procedure to the peak areas of analyte prepared in mobile phase. Blank plasma was obtained from eight healthy volunteers including 2 lipemic blank plasma and 2 hemolyzed blank plasma. For matrix effect analysis in urine, 6 extracts of blank urine were used. For stability analysis, analytes were submitted to 24 h shortterm 8 ◦ C temperature, three freezes–thaw (−70 ◦ C for 24 h and thawed at room temperature) cycles and 24 h in the autoinjector at 12 ◦ C stability tests. The obtained results were compared with those obtained by the analysis of freshly prepared calibration curve and expressed as the RE%.

F.d.L. Moreira, M.P. Marques, G. Duarte et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

5

Fig. 3. Representative chromatograms of raltegravir glucuronide (A) and raltegravir D3 glucuronide (IS) (40 ng/mL in methanol) (B): blank plasma sample (1), LLOQ 2.5 ng/mL (2) and patient plasma sample taken at 2 h after raltegravir administration (3). Retention time of raltegravir glucuronide and raltegravir D3 glucuronide is 4.98 min.

Fig. 4. Representative chromatograms of raltegravir (A) and raltegravir D3 (IS) (40 ng/mL in methanol) (B): blank plasma sample (1), LLOQ 10 ng/mL (2) and patient plasma sample taken at 2 h after raltegravir administration (3). Retention time of raltegravir and raltegravir D3 is 4.66 min.

2.8. Application of the method The protocol was approved by the Research Ethics Committee of the School of Pharmaceutical Sciences of Ribeirão Preto of São Paulo University, Brazil (Protocol no. CEP/FCFRP n 443 – CAAE n 67037617.3.0000.5403). After providing free informed consent, the study enrolled one HIV-infected pregnant woman during the third trimester of pregnancy (33 weeks pregnant) and postpartum (4 weeks after delivery) receiving 400 mg raltegravir twice daily in combination with tenofovir/lamivudine (300 mg/300 mg once daily). Serial blood samples were collected at times 0, 0.5; 1; 2; 3; 4; 6; 8 and 12 h after RAL administration. The urine was collected for 12 h with sampling intervals of approximately 2 h following RAL administration. At the time of delivery, samples of

maternal blood and umbilical cord blood were simultaneously collected to determine the transplacental transfer of RAL and RAL GLU. Plasma aliquots obtained after centrifugation of blood as well as urine samples were stored at −70 ◦ C until the time of analysis. The pharmacokinetic parameters were calculated using the WinNonlin software, version 4.0 (Pharsight Corp, Moutain View, CA, USA) based on the plasma concentration versus time curves. The pharmacokinetic parameters were obtained by a noncompartmental model. The apparent fraction of unchanged RAL eliminated (Fel /F) was calculated by dividing the amount of RAL recovered in 12 -h urine by the RAL dose. The renal clearance was calculated by multiplying the total clearance by the Fel /F. The transplacental transfer was evaluated as the ratio of umbilical cord to maternal plasma concentrations.

6

F.d.L. Moreira, M.P. Marques, G. Duarte et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

Table 2 Validation parameters of the method of analysis of raltegravir in urine.

Table 4 Validation parameters of the method of analysis of raltegravir in plasma.

Validation Parameter

Validation Parameter

Matrix Effect Low QC (0.3 ␮g/mL) High QC (14.6 ␮g/mL) Linearity Linearity (␮g/mL) Linear Equation r2 Precision and Accuracy Intra-assay (n = 5) 0.15 ␮g/mL 0.3 ␮g/mL 9.75 ␮g/mL 14.6 ␮g/mL 130 ␮g/mL (1:40) Inter-assay (n = 15) 0.15 ␮g/mL 0.30 ␮g/mL 9.75 ␮g/mL 14.6 ␮g/mL 130 ␮g/mL (1:40) Stability (n=3) (%RE) Freeze-thaw Shor-term (24 h, 8 ◦ C) Post-processing (24 h)

Matrix Effect Matrix Effect 0.97 0.98

CV (%) 11.3 1.9

0.15 – 19.50 y = 1.4287 x + 0.120756 0.995 Precision [CV(%)] 8.6 2.7 5.2 3.3 2.4

Accuracy [%RE] 2.0 −2.3 1.0 1.1 −0.2

8.4 5.6 6.1 4.9 6.0 Low QC (0.30 ␮g/mL) −1.2 −8.1 5.4

−1.7 0.7 2.5 3.2 2.7 High QC (14.6 ␮g/mL) −0.6 −4.6 −0.7

Table 3 Validation parameters of the method of analysis of raltegravir glucuronide in urine. Validation Parameter

Intra-assay(n = 5) 0.15 ␮g/mL 0.3 ␮g/mL 9.75 ␮g/mL 14.6 ␮g/mL 130 ␮g/mL (1:40) Inter-assay (n=15) 0.15 ␮g/mL 0.30 ␮g/mL 9.75 ␮g/mL 14.6 ␮g/mL 130 ␮g/mL (1:40) Stability (n=3) (%RE) Freeze-thaw Shor-term (24 h, 8◦ C) Post-processing (24 h)

10 ng/mL 20 ng/mL 1000 ng/mL 1600 ng/mL 4000 ng/mL (1:10) Inter-assay (n=15) 10 ng/mL 20 ng/mL 1000 ng/mL 1600 ng/mL 4000 ng/mL (1:10) Stability (n = 3) (% RE) Freeze-thaw Short-term (24 h, 8 ◦ C) Post-processing (24 h)

Matrix effect 1.02 1.05

CV (%) 3.0 1.8

10 – 2000 y = 0.997576 x – 4.16019 0.995 Precision [CV%] 3.1 1.7 2.9 1.9 3.8

Accuracy [% RE] −3.9 −2.5 1.1 −7.7 −4.5

6.4 4.6 3.5 2.9 6.8 Low QC (20 ng/mL) 3.5 −4.7 0.2

−2.3 −3.0 3.6 −4.7 −0.8 High QC (1600 ng/mL) −8.2 −7.6 −1.1

Table 5 Validation parameters of the method of analysis of raltegravir glucuronide in plasma. Validation Parameter

Matriz Effect Low QC (0.3 ␮g/mL) High QC (14.6 ␮g/mL) Linearity Linearity (␮g/mL) Linear Equation r2 Precision and Accuracy

Low QC (20 ng/mL) High QC (1600 ng/mL) Linearity (ng/mL) Linearity (ng/mL) Linear equation r2 Precision and Accuracy Intra-assay (n = 5)

Matriz Effect 0.97 0.98

CV (%) 11.3 1.9

0.15 – 19.50 y = 1.4287 x + 0.120756 0.995 Precision [CV(%)] 8.6 2.7 5.2 3.3 2.4

Accuracy [%RE] 2.0 −2.3 1.0 1.1 −0.2

8.4 5.6 6.1 4.9 6.0 Low QC (0.30 ␮g/mL) −1.2 −8.1 5.4

−1.7 0.7 2.5 3.2 2.7 High QC (14.6 ␮g/mL) −0.6 −4.6 −0.7

Matrix Effect Low QC (5ng/mL) High QC (600 ng/mL) Linearity Linearity (ng/mL) Linear Equation r2 Precision and Accuracy Intra-assay (n = 5) 2.5 ng/mL 5 ng/mL 400 ng/mL 600 ng/mL 2000 ng/mL (1:10) Inter-assay (n=15) 2.5 ng/mL 5 ng/mL 400 ng/mL 600 ng/mL 2000 ng/mL (1:10) Stability (n = 3) (% RE) Freeze-Thaw Short-term (24 h, 8◦ C) Post-processing (24 h)

Matrix effect 0.97 0.96

CV(%) 14.5 4.6

2.5 – 800 y = 0.0361 x + 0.0399 0.994 Precision [CV(%)] 4.6 5.2 7.9 4.2 3.3

Accuracy [%RE] 0.6 6.7 −3.1 −3.1 2.0

6.1 5.2 6.7 5.2 4.1 Low QC (5 ng/mL) 1.9 1.3 −7.6

−4.8 3.6 −0.4 −2.5 0.2 High QC (600 ng/mL) 3.4 3.1 −2.4

3. Results and discussion The initial aim of this study was to develop a method for the simultaneous analysis of RAL and RAL GLU in plasma and urine samples with application in pharmacokinetic studies. However, RAL and RAL GLU generate similar product mass fragments and require separation in the chromatographic system. Furthermore, the analysis of RAL GLU in plasma is subject to matrix interferences and in urine requires lower desolvation temperature when compared to the unchanged RAL. Thus, this study shows the development and validation of 4 different methods for the analysis of RAL and RAL GLU in plasma and in urine samples.

The method for the analysis of RAL and RAL GLU in urine was developed using the Quattro Micro API triple quadrupole system performed in the positive ionization mode. The C18 column (75 × 4.6 mm, 2.7 ␮m) with isocratic elution (acetonitrile containing 0.1% formic acid: water containing 0.1% formic acid, 60:40, v/v) resulted in chromatograms with good peaks resolution. The retention times were approximately 4.96 min and 3.90 min for RAL (Fig. 1) and RAL GLU (Fig. 2), respectively. Other studies also mentioned the in-source transformation phenomenon responsible for the interference of RAL GLU in RAL analysis, showing that the chromatographic separation of both compounds are required

F.d.L. Moreira, M.P. Marques, G. Duarte et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

7

Fig. 5. Representative chromatograms of raltegravir (4000 ng/mL in methanol) (A) and raltegravir glucuronide (400 ng/mL in methanol) (B). Retention time of raltegravir is 4.75 min and raltegravir glucuronide is 3.51 min.

[15,23,25,26]. The desolvation temperature that allowed the highest ionization efficiency of RAL and RAL GLU was 400 ◦ C and 200 ◦ C, respectively. Considering that other desolvation temperatures did not allow a proper ionization of the analytes, RAL and RAL GLU were analyzed separately in urine samples. Few studies showed the validation of an analytical method for RAL GLU quantification in plasma samples [23,25]. Jourdil et al. [25] treated the patient’s plasma with ␤-glucuronidase, an enzyme that catalyzes hydrolysis of the ␤-D-glucuronic acid chemical function, releasing free RAL. Nevertheless, the reaction with ␤-glucuronidase is an additional time-consuming step in sample preparation. Wang et al. [23] developed a LC–MS/MS method for simultaneous determination of RAL and RAL GLU in 50 ␮L of human plasma and employing protein precipitation sample preparation. Compared to this cited method, the present method employed half of plasma volume for the analysis. Furthermore, the extraction procedure also is simple and fast, requiring only the addition of acetonitrile to remove protein in the plasma and urine. For the analysis of RAL and RAL GLU in plasma, a more sensitive instrument was employed (Xevo TQ-S® triple quadrupole mass spectrometer), since the concentration of the analytes in plasma is lower than in urine. The simultaneous determination of RAL and RAL GLU also was not possible because it was observed interference from matrix components co-eluting with RAL GLU. So, RAL GLU was analyzed in plasma samples using a negative mode and a gradient elution in order to eliminate the cited interferences (Fig. 3). Fayet et al. [15] also described the use of negative mode for RAL GLU analysis in plasma samples. Neely et al. [24] reported a method using elution gradient for quantification of RAL and RAL GLU in plasma and urine and employing the negative mode for RAL GLU determination. RAL was determined in plasma samples using the same isocratic conditions employed in urine analysis and eluted approximately at 4.66 min (Fig. 4). The ionization of RAL GLU in positive mode produces the [M+H]+ m/z 445 ion corresponding to RAL. Nevertheless, RAL GLU did not interfere in RAL quantification in plasma, since RAL GLU eluted approximately at 3.51 min (Fig. 5). Validation parameters of the analytical methods for RAL and RAL GLU in urine are presented in Tables 2 and 3, respectively. Validation parameters of the analytical methods for RAL and RAL GLU in plasma are presented in Tables 4 and 5, respectively. The methods showed linearities over the concentration ranges of 10–2000 ng/mL for RAL and 2.5–800 ng/mL for RAL GLU in plasma and 0.1–13.5 ␮g/mL for RAL and 0.15–19.5 ␮g/mL for RAL GLU in urine. A 10-fold dilution of urine with water containing 0.1% formic acid before the extraction was employed in RAL and RAL GLU analysis. Both patient’s urine and blank urine used to construct the

Fig. 6. A) Plasma concentrations of raltegravir (RAL) (solid line) and raltegravir glucuronide (RAL GLU) (dashed line) versus time in a HIV-infected woman during third trimester of pregnancy (filled circles) and postpartum (filled triangles) following RAL doses of 400 mg/12 h. B) Cumulative amount excreted into urine of RAL (solid line) and RAL GLU (dashed line) in a HIV-infected woman during third trimester of pregnancy (filled circles) and postpartum (filled triangles).

calibration curve were diluted before the sample preparation step. The formic acid improved the ionization of the analytes during analysis in urine. Neely et al. [24] also reported a method employing a 20-fold dilution of urine. Due the high inter- and intra-patient variability [10] in RAL and RAL GLU concentrations in biological matrices, some samples needed to be diluted prior to analysis. The dilution QC results demonstrated acceptable accuracy and precision in RAL and RAL GLU quantification in urine and human plasma. The intra- and inter-assay precision and accuracy of plasma and urine QC samples for RAL and RAL GLU met the acceptance criteria. No significant interference was observed in the analytes retention

8

F.d.L. Moreira, M.P. Marques, G. Duarte et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

Table 6 Pharmacokinetic parameters and transplacental transfer data of raltegravir (RAL) and raltegravir glucuronide (RAL GLU) in a HIV-infected woman receiving RAL 400 mg twice daily. RAL

RAL GLU

Parameter

third trimester

postpartum

third trimester

postpartum

Cmax (␮g/mL) Tmax (h) C12h (␮g/mL) AUC0-12h (h*␮g/mL) CLss /F (L/h) Vd /F (L/h) Ae 0-12h (mg) Fe /F% CLr (L/h) Umbilical cord/maternal plasma ratio*

1.5 4 0.2 8.0 45.9 167.9 34.2 8.6 3.9 0.7

2.1 6 0.1 7.6 51.1 99.8 7.5 1.9 1.0

2.2 6 0.7 14.2 – – 114.2 – – 0.8

3.5 6 0.4 19.7 – – 39.8 – –

Cmax , maximum plasma concentration; tmax , time to reach Cmax ; AUC0− ∞ , area under the plasma concentration vs time curve; C12h ; concentration 12 h after last dose; Vd /F, apparent volume of distribution; CLss /F, apparent clearance; Ae 0–12 h , amount excreted into urine from 0 to 12 h; Fel/F, apparent elimination fraction; CLr /F apparent renal clearance. * the interval between maternal dosing and delivery is 1.83 h. -: not determined.

time during injection of blank biological matrix (plasma or urine). Furthermore, it was not observed carry-over effects after injection of blank biological matrix sample following an injection of ULOQ. No significant matrix effect was observed in RAL and RAL GLU analysis in plasma or urine and all CVs were < 15%. Stability of the plasma and urine samples of RAL and RAL GLU was confirmed by short-term (24 h at +8 ◦ C), post-processing (24 h at 12 ◦ C) tests and after three freeze–thaw cycles, when compared with freshly prepared samples. The present methods were applied in a maternal-fetal pharmacokinetic study of RAL and RAL GLU in a HIV-infected woman receiving RAL 400 mg twice daily. The plasma and urine concentration-time curves of RAL and RAL GLU during pregnancy and postpartum are demonstrated in Fig. 6 and Table 6. The pharmacokinetic parameters obtained during third trimester of pregnancy and postpartum of RAL and RAL GLU are comparable to obtained in healthy volunteers [24]. Furthermore, the plasma pharmacokinetic parameters obtained during third trimester of pregnancy of RAL are similar with values demonstrated in other studies in HIV-infected pregnant women [13,14]. This is the first report of pharmacokinetic data of RAL in urine and RAL GLU in plasma and urine in a HIV-infected pregnant woman. The transplacental transfer of RAL and RAL GLU is 0.72 and 0.84, respectively. The transplacental transfer of RAL GLU is closely to RAL. 4. Conclusion Precise and accurate LC–MS/MS methods for determination of RAL and RAL GLU in urine and plasma have been developed and validated. The extraction procedure is simple and fast. This is, to the best of our knowledge, the first report of the validated methods for quantification of RAL and RAL GLU in urine. The methods have been successfully applied in a maternal-fetal pharmacokinetic study. Declaration of Competing Interest The authors declare no conflict of interest.

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12] [13]

[14]

Acknowledgments The authors thank the Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support (grant number FAPESP 2016/ 23938-2 and 2018/05616-3).

[15]

References

[16]

[1] Ministério da Saúde, Protocolo Clínico e Diretrizes Terapêuticas para Prevenc¸ão da Transmissão Vertical de HIV, Sífilis e Hepatites Virais, 2018, pp.

1–252 (Accessed June 2019) http://www.aids.gov.br/pt-br/pub/2015/ protocolo-clinico-e-diretrizes-terapeuticas-para-prevencao-da-transmissaovertical-de-hiv. Department of Health and Human Services, Recommendations for the Use of Antiretroviral Drugs in Pregnant Women with HIV Infection and Interventions to Reduce Perinatal HIV Transmission in the United States, 2018, pp. 1–366 (Accessed June 2019) https://aidsinfo.nih.gov/contentfiles/ lvguidelines/perinatalgl.pdf. European AIDS Clinical Society, European Guidelines for Treatment of HIV Infected Adults in Europe, 2017, pp. 1–102 (Accessed June 2019) http://www. eacsociety.org/files/guidelines 9.0-english.pdf. L. Croci, M. Trezzi, M.P. Allegri, T. Carli, S. Chigiotti, M.P. Riccardi, B. Ricciardi, M. Toti, C. Nencioni, Pharmacokinetic and safety of raltegravir in pregnancy, Eur. J. Clin. Pharmacol. 68 (2012) 1231–1232. A. Maliakkal, S. Walmsley, A. Tseng, Review of the efficacy, safety, and pharmacokinetics of Raltegravir in pregnancy, J. Acquir. Immune Defic. Syndr. 72 (2016) 153–161. A. Hegazi, P. Hay, HIV seroconversion in the third trimester of pregnancy: using raltegravir to prevent mother-to-child transmission, Int. J. STD AIDS 24 (2013) 245–246. K. Kassahun, I. Mcintosh, D. Cui, D. Hreniuk, S. Merschman, K. Lasseter, N. Azrolan, M. Iwamoto, J.A. Wagner, L.A. Wenning, Metabolism and disposition in humans of raltegravir (MK-0518), an Anti-AIDS drug targeting the human immunodeficiency virus 1 integrase enzyme, Drug Metab. Dispos. 35 (2007) 1657–1663. L. Belkhir, C. Seguin-Devaux, L. Elens, C. Pauly, N. Gengler, S. Schneider, J. Ruelle, V. Haufroid, B. Vandercam, Impact of UGT1A1 polymorphisms on Raltegravir and its glucuronide plasma concentrations in a cohort of HIV-1 infected patients, Sci. Rep. 8 (2018) 7359. B. Hernández-Novoa, A. Moreno, M.J. Pérez-Elías, C. Quereda, F. Dronda, J.L. Casado, N. Madrid-Elena, M. Aguilar, E. Fumero, J. Moltó, S. Moreno, Raltegravir pharmacokinetics in HIV/HCV-coinfected patients with advanced liver cirrhosis (Child-Pugh C), J. Antimicrob. Chemother. 69 (2014) 471–475. D. Cattaneo, C. Gervasoni, P. Meravigilia, S. Landonio, S. Fucile, V. Cozzi, S. Baldelli, M. Pellegrini, M. Galli, E. Clementi, Inter- and intra-patient variability of raltegravir pharmacokinetics in HIV-1-infected subjects, J. Antimicrob. Chemother. 67 (2012) 460–464. M. Arab-alameddine, A. Fayet-Mello, R. Lubomirov, M. Neely, J. di Iulio, A. Owen, M. Boffito, M. Cavassini, H.F. Günthard, K. Rentsch, T. Buclin, M. Aouri, A. Telenti, L.A. Decosterd, M. Rotger, C. Csajka, Population pharmacokinetic analysis and pharmacogenetics of raltegravir in HIV-Positive and healthy individuals, Antimicrob. Agents Chemother. 56 (2012) 2959–2966. D.M. Burger, Raltegravir: a review of its pharmacokinetics, pharmacology and clinical studies, Expert Opin. Drug Metab. Toxicol. 6 (2010) 1151–1160. D.H. Watts, A. Stek, B.M. Best, J. Wang, E.V. Capparelli, T.R. Cressey, F. Aweeka, P. Lizak, R. Kreitchmann, S.K. Burchett, D.E. Shapiro, E. Hawkins, E. Smith, M. Mirochnick, Raltegravir pharmacokinetics during pregnancy, J. Acquir. Immune Defic. Syndr. 67 (2014) 375–381. M. Blonk, A. Colbers, C. Hidalgo-Tenorio, K. Kabeya, K. Weizsäcker, A.E. Haberl, J. Moltó, D.A. Hawkins, M.E. van der Ende, A. Gingelmaier, G.P. Taylor, J. Ivanovic, C. Giaquinto, D.M. Burger, Raltegravir in HIV-1 infected pregnant women: pharmacokinetics, safety and efficacy, Clin. Infect. Dis. 61 (2015) 809–816. A. Fayet, A. Béguin, B. Zanolari, C. Cruchon, N. Guignard, A. Telenti, M. Cavassini, H.F. Günthard, T. Buclin, J. Biollaz, B. Rochat, L.A. Decosterd, A LC–tandem MS assay for the simultaneous measurement of new antiretroviral agents: raltegravir, maraviroc, darunavir, and etravirine, J. Chromatogr. B 877 (2009) 1057–1069. K. Tsuchiya, M. Ohuchi, N. Yamane, H. Aikawa, H. Gatanaga, S. Oka, A. Hamada, High-performance liquid chromatography–tandem mass spectrometry for simultaneous determination of raltegravir, dolutegravir and

F.d.L. Moreira, M.P. Marques, G. Duarte et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112838

[17]

[18]

[19]

[20]

[21]

elvitegravir concentrations in human plasma and cerebrospinal fluid samples, Biomed. Chromatogr. 32 (2018) 4058. Z. Djerada, C. Feliu, C. Tournois, D. Vautier, L. Binet, A. Robinet, H. Marty, C. Gozalo, D. Lamiable, H. Millart, Validation of a fast method for quantitative analysis of elvitegravir, raltegravir, maraviroc, etravirine, tenofovir, boceprevir and 10 other antiretroviral agents in human plasma samples with a new UPLC-MS/MS technology, J. Pharm. Biomed. Anal. 86 (2013) 100–111. R. Ter Heine, J.W. Mulder, E.C. van Gorp, J.F. Wagenaar, J.H. Beijnen, A.D. Huitema, Clinical evaluation of the determination of plasma concentrations of darunavir, etravirine, raltegravir and ritonavir in dried blood spot samples, Bioanalysis 3 (2011) 1093–1097. A.K. Blakney, Y. Jiang, D. Whittington, K.A. Woodrow, Simultaneous measurement of etravirine, maraviroc and raltegravir in pigtail macaque plasma, vaginal secretions and vaginal tissue using a LC-MS/MS assay, J. Chromatogr. B 1025 (2016) 110–118. S. Quaranta, C. Woloch, A. Paccou, M. Giocanti, C. Solas, B. Lacarelle, Validation of an electrospray ionization LC-MS/MS method for quantitative analysis of raltegravir, etravirine, and 9 other antiretroviral agents in human plasma samples, Ther. Drug Monit. 31 (2009) 695–702. E. Yamada, R. Takagi, K. Sudo, S. Kato, Determination of abacavir, tenofovir, darunavir, and raltegravir in human plasma and saliva using liquid chromatography coupled with tandem mass spectrometry, J. Pharm. Biomed. Anal. 114 (2015) 390–397.

9

[22] T.L. Parsons, M.A. Marzinke, Development and validation of a liquid chromatographic-tandem mass spectrometric method for the multiplexed quantification of etravirine, maraviroc, raltegravir, and rilpivirine in human plasma and tissue, J. Pharm. Biomed. Anal. 131 (2016) 333–344. [23] L.-Z. Wang, L.S. Lee, W.-L. Thuya, G.H. Soon, L.-R. Kong, P.-L. Nye, E.J.D. Lee, C. Flexner, B.-C. Goh, Simultaneous determination of raltegravir and raltegravir glucuronide in human plasma by liquid chromatography–tandem mass spectrometric method, J. Mass Spectrom. 46 (2011) 202–208. [24] M. Neely, L. Decostered, A. Fayet, J.S.F. Lee, A. Margol, M. Kanani, J. Iulio, T. Schoen-Angerer, R. Jellife, A. Calmy, Pharmacokinetics and Pharmacogenomics of once-daily raltegravir and atazanavir in healthy volunteers, Antimicrob. Agents Chemother. 54 (2010) 4619–4625. [25] J.F. Jourdil, M. Bartoli, F. Stanke-Labesque, Lack of specificity for the analysis of raltegravir using online sample clean-up liquid chromatography–electrospray tandem mass spectrometry, J. Chromatogr. B 877 (2009) 3734–3738. [26] R. Ter Heine, M.J. Hillebrand, H. Rosing, E.C. van Gorp, J.W. Mulder, J.H. Beijnen, A.D. Huitema, Quantification of the HIV-integrase inhibitor raltegravir and detection of its main metabolite in human plasma, dried blood spots and peripheral blood mononuclear cell lysate by means of high-performance liquid chromatography tandem mass spectrometry, J. Pharm. Biomed. Anal. 49 (2009) 451–458.