Sensitive and Rapid LCMS/MS Method for the Estimation of recently approved Antiviral drugs Maribavir and Fostemsavir in spiked human plasma

 

Penchala Reddy Vaka, Battula Sreenivasa Rao*

Department of Chemistry, GITAM Institute of Science, GITAM (Deemed to be University)

Visakhapatnam – 530045.

*Corresponding Author E-mail: sbattula2@gitam.edu

 

ABSTRACT:

In this study, a straightforward, highly sensitive, and selective liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and rigorously validated for the simultaneous quantification of Maribavir and Fostemsavir in human plasma. To ensure precision and reliability, we employed Dolutegravir as the internal standard (IS). The analytical process involved a two-step extraction method. Initially, protein precipitation was induced by the addition of acetonitrile, followed by liquid–liquid extraction using a 1:1 (v/v) mixture of diethyl ether and dichloromethane as the extracting solvent. Separation of the analytes was achieved through reversed phase high-performance liquid chromatography (HPLC) using a Phenomenex C18 Luna column (4.6 mm×100 mm, 5 µm). A simple isocratic mobile phase consisting of acetonitrile, methanol, and 0.1% formic acid (35:55:10, v/v) was used, operating at a flow rate of 0.5 mL/min. Under these optimized conditions, the LC chromatogram of the spiked standard exhibited distinct peaks at retention times of 2.07 min, 2.59 min, and 4.29 min for Fostemsavir, Maribavir, and the internal standard, respectively. Detection was performed using a triple quadrupole mass spectrometer employing electrospray ionization in positive ion mode and multiple reaction monitoring (MRM) mode. The mass transitions monitored were m/z 377 → 110, m/z 584 → 105, and m/z 420 → 142 for maribavir, fostemsavir, and dolutegravir, respectively. This method provided a rapid analysis within 5 minutes, over a linear concentration range of 15-750 ng/mL for both maribavir and fostemsavir. Method validation was conducted following FDA guidelines for bio-analytical methods, and the results consistently fell within the acceptable limits for both analytes. Therefore, our developed method holds promise for the accurate analysis of maribavir and fostemsavir in human plasma, and it has potential applications in pharmacokinetic studies

 

KEYWORDS: Maribavir, Fostemsavir, LC–MS/MS, Human Plasma, Internal standard, Dolutegravir.

 

 

INTRODUCTION: 

Maribavir is a cytomegalovirus (CMV) pUL97 kinase inhibitor and antiviral class medical drug prescribed for the treatment of treat post-transplant cytomegalovirus1. It is indicated for the treatment of paediatric patients (weighing >35kg and at least 12 years old) and adult with post-transplant CMV infection which is refractory to standard treatment with foscarnet, cidofovir, ganciclovir and valganciclovir. Maribavir blocks the virus replication by inhibiting the activity of human CMV pUL97 enzyme.

 

Fatigue, diarrhea, nausea, vomiting and taste disturbance are common side effects of Maribavir2.

 

Fostemsavir is an antiretroviral class medical drug prescribed to treat adult patients with HIV/AIDS who undergone multiple HIV medications and whose HIV infection cannot be treated with other therapies due to intolerance, safety and resistance considerations3. Fostemsavir works by preventing the CD4 cell connection with virus by binding gp120 protein. Abdominal pain, fatigue, rash, immune reconstitution inflammatory syndrome, vomiting, sleep disturbance, headache, nausea, diarrhoea, indigestion/heartburn and drowsiness are the possible side effects of Fostemsavir4.

 

 

The existing literature review reveals that there is only one reported HPLC method for quantifying fostemsavir in pharmaceutical formulations5. Furthermore, no analytical method established for determination of maribavir and fostemsavir in biological samples. Therefore, this study proposed to develop a straightforward and robust LCMS method for quantifying maribavir and fostemsavir in human plasma. As part of this study, dolutegravir, an antiviral drug, was finalized as internal standard. The chemical structures of maribavir, fostemsavir, and the internal standard dolutegravir are depicted in Figure 1.

 

 

A

 

 

B

 

 

C

Figure 1: Structure of maribavir (A), fostemsavir (B) and dolutegravir internal standard (C)

 

MATERIALS AND METHODS:

Chemicals and Equipment:

The analytical standard maribavir was sourced from Takeda Pharmaceuticals India Pvt Ltd., located in Haryana, while the standard drugs fostemsavir and dolutegravir were obtained from GSK Pharmaceutical Ltd., located in Secunderabad, Telangana. Procured HPLC-grade methanol, acetonitrile, and Milli-Q water from Merck Chemicals in Mumbai. Healthy human blood was obtained from a nearby diagnostic laboratory, and plasma was isolated from the whole blood by centrifugation, using a Pasteur pipette. The experimental analysis was conducted using a Waters (Japan) Alliance 2695 LCMS system, which was coupled with a triple quadrupole mass detector known as Waters ZQ (LAA 1369). The system was equipped with an auto-injector capable of handling volumes ranging from 0.1 to 1500 µL, and data integration was performed using MassLynx 4.2 software by Waters. The analytes were separated using a Phenomenex C18 Luna column measuring 4.6 mm in diameter and 100mm in length, with a particle size of 5µm

 

Plasma Extraction:

The extraction of the drug from spiked plasma samples involved a two-step process, beginning with protein precipitation and liquid-liquid extraction. Initially, 1mL of acetonitrile solvent was added. Subsequently, the mixture was thoroughly agitated for 2minutes. Liquid-liquid extraction was carried out using a 1:1 (v/v) ratio of diethyl ether and dichloromethane, totalling 3 mL of the solvent mixture. Then it was mixed vigorously and centrifuged for 5min at 4000rpm. The supernatant decanted carefully, dried and then resulting dried residue was reconstituted with 1mL of methanol. 

 

Method development:

The LCMS method for separation and quantification of Fostemsavir and Maribavir was meticulously developed through a systematic optimization of multiple analytical parameters. Key factors including the composition and pH of the mobile phase, the flow rate, the choice of stationary phase, and the mass spectrometer operating conditions were thoroughly investigated. The analytical conditions that yielded the most favourable chromatographic outcomes, characterized by exceptional system suitability, were identified as the optimal parameters for the analytical method. Subsequently, these selected conditions underwent a comprehensive validation process to ensure their reliability and accuracy.

 

Method validation:

The validation of the developed method for the analysis of Fostemsavir and Maribavir, employing Dolutegravir as an internal standard, adhered to the method validation guidelines outlined by FDA6 and the guidance available in existing literature7-19. A comprehensive validation procedure was executed, encompassing an evaluation of various critical parameters, namely selectivity, sensitivity, linearity, recovery, matrix effect, accuracy, and precision. To assess the robustness and reliability of the analytical method, an extensive battery of stability studies was conducted. Importantly, these assessments were carried out at Low Quality Control (LQC), Medium Quality Control (MQC), and High Quality Control (HQC) levels, all falling within the predefined calibration range

 

RESULTS:

This study focused on the development of a suitable LCMS analytical method for the effective separation and analysis of Fostemsavir and Maribavir, with Dolutegravir serving as the internal standard. The method development process was successfully concluded by achieving optimal chromatographic results. The separation was attained using a mobile phase consisting of acetonitrile, methanol, and 0.1% formic acid in a ratio of 35:55:10 (v/v). The mobile phase operated at a flow rate of 0.5 mL/min, which contributed to the economical usage of the mobile phase. A minimal sample volume of 2 µL was injected and separated on a Phenomenex C18 Luna column with dimensions of 4.6 mm x 100 mm and a particle size of 5 µm. The column eluents were simultaneously monitored using both UV and mass detectors at room temperature.

 

In the developed method conditions, the chromatogram obtained for the blank (un-spiked sample) showed no discernible peaks throughout the entire runtime. However, in the chromatogram of the spiked standard, distinct peaks were observed at retention times of 2.07 minutes, 2.59 minutes, and 4.29 minutes, corresponding to Fostemsavir, Maribavir, and the internal standard, respectively. The peak area responses and elution times for each individual analyte in their respective analyses were found to be consistent with the results obtained from the combined spiked sample chromatogram. This confirmed the method's specificity for the precise analysis of the target analytes in this study. Fig. 2A, 2B, 2C and 2D shows the system suitability and specificity chromatograms observed in the developed method conditions.


 

A) blank plasma; B) maribavir spiked plasma; C) fostemsavir spiked plasma; D) internal standard (dolutegravir) spiked plasma; E) analytes and internal standard spiked plasma

Figure 2: System suitability chromatograms observed in the study

 

The full scan Q1 mass spectra revealed predominant protonated [M + H]+ parent ions at m/z values of 377, 584, and 420 for maribavir, fostemsavir, and dolutegravir, respectively. Additionally, characteristic and highly abundant fragment ions were observed at m/z 245, 105 and 142, corresponding to maribavir, fostemsavir, and dolutegravir, respectively, in the product ion mass spectrum. A summary of the mass spectral parameters is presented in Table 1, and Figure 3 displays the full scan mass spectra observed for the analytes in this study.

 

Table 1: Optimized LC–MS/MS conditions optimized in the study

S No

Parameter

Results

Fostemsavir

Maribavir

Dolutegravir

1

Mass of Precursor (m/z)

584

377

420

2

Mass of product ion (m/z)

105

245

142

3

Cell exit potential (v)

16

24

15

4

Collision energy (v)

25

31

28

5

Entrance potential (v)

15

11

14

6

Declustering potential (v)

32

44

32

 

 

Figure 3: Mass fragmentation spectra of analytes in the study

A) Maribavir; B) Fostemsavir; C) Dolutegravir internal standard

 

The developed method exhibited a detection limit of 3.0 ng/mL for both fostemsavir and maribavir, while the quantification limit was established at 10ng/mL. This underscored the method's remarkable sensitivity, capable of detecting concentrations as low as 10ng/mL. Calibration dilutions spanning from the limit of quantification (LOQ) concentration to a considerably higher range were meticulously prepared. The regression analysis of these calibration dilutions yielded highly accurate and strongly correlated calibration curves. For both analytes, fostemsavir and maribavir, the calibration curve covered the concentration range of 10 to 750 ng/mL, with regression equations of y = 0.0052x+ 0.0302 (R˛ = 0.9994) and y = 0.0046x+0.1109 (R˛ = 0.9997), respectively.

 

The precision of the LCMS-based method developed for the analysis of fostemsavir and maribavir was rigorously assessed through a precision study. This study encompassed High Quality Control (HQC), Medium Quality Control (MQC), and Low Quality Control (LQC) levels, spanning the calibration range for both analytes. The % accuracy obtained within the intraday precision ranged from 99.71% to 99.83% for fostemsavir, while for maribavir, it fell within the range of 96.38% to 99.91%. In the interday precision assessment, fostemsavir exhibited % accuracy in the range of 96.88% to 99.95%, whereas maribavir ranged from 95.02% to 97.00%. These % accuracy values comfortably met the acceptable limits. Furthermore, the % Relative Standard Deviation (%RSD) for the repeated analysis results was found to be well within the acceptable limit of less than 2% for both fostemsavir and maribavir. Detailed findings from the study are presented in Table 2. In light of these results, it can be confidently concluded that the method is characterized by exceptional precision and accuracy.

 

Table 2: Intra and interday precision and accuracy results for fostemsavir and maribavir in the developed method

Analyte

QC level

Intraday precision (n=6)

Interday precision (n=6)

Conc.

found (ng/mL)

Accuracy %

RSD %

Conc.

found (ng/mL)

Accuracy %

RSD %

Fostemsavir

HQC (750 ng/mL)

748.7

99.83

0.28

726.6

96.88

0.93

MQC (300 ng/mL)

299.16

99.72

0.41

299.86

99.95

0.34

LQC (10 ng/mL)

9.97

99.71

0.59

9.80

97.95

0.69

Maribavir

HQC (750 ng/mL)

749.3

99.91

0.15

724.2

96.56

1.18

MQC (300 ng/mL)

289.15

96.38

1.27

290.99

97.00

0.65

LQC (10 ng/mL)

9.76

97.62

0.81

9.50

95.02

0.83

The efficiency of analyte extraction in the developed method was comprehensively assessed through recovery studies for both fostemsavir and maribavir. In these studies, the %accuracy of spiked samples was determined by comparing the peak area ratios of spiked samples with the aqueous calibration curve at corresponding concentration levels within the calibration range. This assessment covered High Quality Control (HQC), Medium Quality Control (MQC), and Low Quality Control (LQC) samples. The % recovery values obtained ranged from 95.91% to 101.25% for fostemsavir and from 97.57% to 102.39% for maribavir within the developed method. These results confirm that the method exhibits highly efficient extraction capabilities, signifying its accuracy and recoverability.

 

The stability of the analytes over various storage time intervals was meticulously examined through short-term and long-term stability assessments. Freeze-thaw stability was evaluated to ascertain the impact of three freeze-thaw cycles on analyte stability, while auto-sampler stability was assessed to ensure analyte stability during storage in the auto-sampler. Throughout these stability studies, which encompassed Low, Medium, and High Quality Control samples, % stability was calculated by comparing the results with the corresponding standard calibration curve. The outcomes of the stability studies, including short-term, long-term, freeze-thaw, auto-sampler, and dry extract stability, consistently demonstrated that the mean % nominal values of the analytes fell within ±15% of the predicted concentrations for the LQC, HQC, and MQC levels. The detailed results of the stability studies are presented in Table 3, confirming that the analytes exhibited acceptable stability limits. These findings underscore the method's robust stability and its suitability for the analysis of fostemsavir and maribavir.


 

Table 3: Results observed in various stability studies conducted for fostemsavir and maribavir in the developed method

S No

Test

QC level

Fostemsavir

Maribavir

Conc.

found (ng/mL)

% Stability

RSD %

Conc.

found (ng/mL)

% Stability

RSD %

1

Short term stability

HQC (750ng/mL)

744.4

99.26

0.98

746.3

99.50

0.56

2

MQC (300ng/mL)

297.43

99.14

0.58

298.14

99.38

0.55

3

LQC (10ng/mL)

9.90

99.03

1.04

9.65

96.54

0.68

4

Long term stability

HQC (750ng/mL)

741.1

98.81

0.47

742.0

98.93

0.70

5

MQC (300ng/mL)

295.96

98.65

0.71

296.42

98.81

0.81

6

LQC (10ng/mL)

9.76

97.61

1.05

9.51

95.09

0.68

7

Freeze–thaw stability

HQC (750ng/mL)

723.6

96.48

1.29

729.5

97.27

0.51

8

MQC (300ng/mL)

286.05

95.35

1.37

287.64

95.88

0.83

9

LQC (10ng/mL)

9.59

95.95

1.64

9.36

93.61

0.78

10

Auto-sampler stability

HQC (750ng/mL)

744.4

99.26

0.38

745.9

99.45

0.22

11

MQC (300ng/mL)

286.01

95.34

1.89

287.40

95.80

0.68

12

LQC (10ng/mL)

9.76

97.57

1.11

9.50

94.96

0.67

13

Dry extract stability

HQC (750ng/mL)

711.5

94.86

1.48

717.3

95.65

1.22

14

MQC (300ng/mL)

285.76

95.25

1.51

285.75

95.25

0.88

15

LQC (10ng/mL)

9.74

97.44

1.23

9.46

94.64

1.09

 

DISCUSSION:

In the extraction of analytes from biological samples, both solid-phase as well as liquid-liquid extraction methods were extensively explored. While solid-phase extraction proved challenging for extracting highly polar compounds and was considered costly, liquid-liquid extraction was deemed simpler and more cost-effective. Consequently, the liquid-liquid extraction was employed for extracting fostemsavir and maribavir, along with the internal standard. Among the solvents tested, the one that yielded the highest chromatographic response was finalized as the most appropriate for analyte extraction. It was determined that an equal-volume mixture of diethyl ether and dichloromethane exhibited the highest peak area responses for fostemsavir, maribavir, and the internal standard, without chromatographic interference and significantly reduced noise. Consequently, this solvent mixture was chosen for analyte extraction from the plasma matrix, and the extracted samples were utilized in the subsequent method development and validation using LCMS.

 

In the choice of the mobile phase, different compositions of acetonitrile and methanol were examined as organic modifiers, and various compositions and pH ranges of 0.01% ammonia were studied. Results indicated that the inclusion of acetonitrile in the mobile phase led to superior separation compared to acetonitrile alone. Under the developed method conditions, the chromatogram for the blank (un-spiked) samples showed no peaks throughout the entire runtime. In contrast, the chromatogram for the spiked standard samples displayed distinct peaks at retention times of 2.07 minutes, 2.59 minutes, and 4.29 minutes, corresponding to fostemsavir, maribavir, and the internal standard. No interference from impurities was detected during the chromatographic runtime, and clear mass fragmentation patterns were identified for each individual analyte, affirming the method's specificity with no matrix effect.

The method exhibited a highly sensitive detection limit of 3.0 ng/mL for both analytes, with a quantification limit of 10 ng/mL. The calibration curve demonstrated linearity across the concentration range from the limit of quantification (LOQ) to 750 ng/mL, with correlation coefficients exceeding 0.999 for both analytes. Method validation results were within acceptable limits, confirming the method's suitability for the precise analysis of fostemsavir and maribavir in biological samples.

 

CONCLUSION:

In summary, this study has introduced a straightforward and robust HPLC–MS/MS method for the simultaneous separation and quantification of fostemsavir and maribavir, in spiked human plasma. The method involves a simple protein precipitation step followed by liquid-liquid extraction of the analytes from the spiked plasma matrix. Notably, the method exhibited a high level of sensitivity, capable of detecting the analytes at concentrations as low as 3.0 ng/mL. Furthermore, it demonstrated excellent linearity, with a well-fitted linear calibration curve spanning the concentration range from the Limit of Quantification (LOQ) of 10 ng/mL to 750 ng/mL. The method underwent a rigorous validation process, meeting all predefined criteria and proving its validity across various parameters. Based on the findings from this study, it can be confidently concluded that the method is well-suited for the simultaneous separation and analysis of fostemsavir and maribavir. Moreover, it holds promise for potential applications in pharmacokinetic profiling of these studied drugs.

 

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Received on 26.04.2022            Modified on 24.03.2023

Accepted on 26.10.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(11):5149-5154.

DOI: 10.52711/0974-360X.2023.00834