Introduction

Tenofovir disoproxil fumarate is utilized in the treatment of Acquired Immuno Deficiency Syndrome (Vergori et al., 2020; Yang et al., 2019) and chronic serum hepatitis. From the age of 2 it is used for HIV-1 treatment by combining with ART (antiretroviral). From the age of 12, it is used to treat chronic serum hepatitis (Nicolò et al., 2015). Tenofovir disoproxil is a nucleotide analogue reverse-transcriptase inhibitor (NtRTI). It is so-called as pro-drug. Concentrations in blood plasma in abstaining people is 25% after an hour and in fatty food condition, it is 40% subsequently two hours. Its hunks the efficiency of reverse transcriptase, which prevents from replicating. It results in a decrease in the content of virus in plasma. It is usually obtained in two forms as a tablet and as powder. Its melting point is 279°C. Tenofovir disoproxil fumarate is a white to off-white crystal-like powder, i.e., dissolvable in methyl alcohol, a little dissolve in H₂O and very little dissolvable in methylene chloride. Tenofovir eliminates waste through kidneys. It acts as a substrate analogue in cellular pigments. Tenofovir is detected in body fluids by liquid chromatography.

Pioglitazone is utilized for non-insulin-dependent diabetes type, which is Glucophage. Subsequent control of pioglitazone reduces insulin protest in the boundary and liver ensuing improvement in insulin subject to dextrose deed and reduces hepatic dextrose outcome. It is utilized together in monotherapy and in grouping with insulin in handling non-insulin-dependent diabetes type. Pioglitazone is also known as 5-[[4-[2-(5-ethyl pyridine-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione;hydrochloride. Its molecular formula is C₁₉H₂₁ClN₂O₃S.

It is utilized to regulate hyperglycaemia and is used to avoid kidney harm, sightless, nervous difficulties and amputate difficulties. Controlling diabetes reduces myocardial infarction. It is used alone or together with other diabetes mellitus pills. Pioglitazone is utilized with proper diet and physical activities and from time to time with extra medicines which are used to treat diabetes type 2 (Balaji, 2013; Hanefeld, 2007). Pioglitazone is a group of glitazones. It takes action to increase the ability to insulin, an organic element that serves to maintain the levels of sugar. It is not utilized for the treatment of diabetes type 1 and ketoacidosis.

Structure of Tenofovir Disoproxil Fumarate is shown in Figure 1 and Pioglitazone HCL is shown in Figure 2.

According to literature survey several techniques such as RP-HPLC (Panchagiri, Begum, Valupadas, & Ciddi, 2018; Reddy & Reddy, 2015), LC-MS (Takahashi et al., 2007; Yadav, Mishra, Singhal, Goswami, & Shrivastav, 2009) were used for the assessment of Tenofovir disoproxil fumarate and to assess Pioglitazone HPLC (Barkil, Gagnieu, & Guitton, 2007; Souri, Jalalizadeh, & Saremi, 2008), HPLC-UV (Elrefay, Omnia, Ismaiel, Hassan, & Shalaby, 2013; Tahmasebi, Yamini, & Saleh, 2009), RP-HPLC (Ramathilagam & Solairaj, 2014; Sultana, Shafi, Arayne, Siddiqui, & Hussain, 2011), C-MS/MS (Jagadeesh, Bharathi, Pankaj, Narayana, & Venkateswarulu, 2013) were used. Our present work determines the simultaneous estimation of Tenofovir disoproxil fumarate and Pioglitazone HCL by LC-MS using human plasma. Parameters such as linearity, LOQ, accuracy and precision, Recovery and sustainability/stability are validated.

EXPERIMENTAL

Chemicals and reagents

Working Standard of Tenofovir disoproxil fumarate and Pioglitazone was obtained from by Akshaya labs, Hyderabad, India. Tablets were acquired from the limited marketplace. Acetonitrile of HPLC grade by Merck, Ammonium Acetate AR grade obtained from Qualigens fine chemicals and Water HPLC grade from Milli-Q RO system was used. HPLC grade reagents were utilized.

LC-MS/MS Instrumentation and Chromatographic Methods

In positive ion mode, the LC-MS/MS instrument was functioned and equipped with ESI using nebulizer, curtain and collision gas which is embraced with AB Sciex API-4000 mass spectrometer. The HPLC setup contains online DGU-20A3 soluble degasser, segment boiler CTO20A, LC20AD drives-2, SIL-HTc sampler. C18-XBridge segment was split into 60:40 proportion with a stream rate of 1.0mL/min kept at 400C. Changeable phase contains 0.1% methanoic acid(A) and ethanenitrile (B). In the primary phase up to 0.8min it is arranged as 95% A/5%B then it gets changed to 5 % A/95% B for 2.2 min. Later it slowly stimulated to 95% A/5% B by 2.4 min and remained the same at 3.5 min.

Optimization of MS/MS detection conditions and parameters

Multi-reaction Monitoring settings were determined by using 500ng/mL of an analyte by distillation. Table 1 shows the optimum MS-parameters of Tenofovir Disoproxil fumarate and Pioglitazone HCL in positive ion mode.

Preparation of stock solution

By using Acetonitrile, 100mg of Tenofovir Disoproxil fumarate and Pioglitazone HCL were dissolved and the final volume is made with H₂O and Acetonitrile in 1:1 ratio to give 1mg/ml. The standard stock solution is stored at -2to ± 20 C till analysis is completed.

Selection of HPLC column

In HPLC column inverted C-18 columns were regularly used for the objective analyte. Primarily sio₂ based C-8 columns are verified and these results in poor separation. Several aspects were taken, such as the composition of the mobile phase and PH stages were examined, yet no enhancement in chromatographic condition. C18 results in fine resolution but presents the uneven peaks. Finally, Phenomenex Luna C18 showed substantially good resolution, symmetrical peaks and less co-elution between target compounds due to its effectiveness in polarity and aromatic selectivity.

Internal standard selection

For positive mode, analysis metoprolol is used. It requires acidic PH for elution but Tenofovir disoproxil fumarate and Pioglitazone requires neutral PH after extraction, which is equity diluted with H₂O for supernate. Finally, it provides good resolution and elution.

Results and Discussion

Validation

It is a secondary phase. It is a very hard and important phase in quantitation and produces absolute results. The guidelines were provided by the FDA to fulfil the essentials requires for the technique. If the guidelines were not obeyed, then it seems to the improper analysis and not used for assent. To regulate the concentration, the procedure starts with a standard curve, which is built form the quantitative analysis standard ranges form 1-1000ng/mL.

The standards were divided in the presence of Metoprolol as internal standard from Tenofovir disoproxil fumarate and Pioglitazone by LC-MS/MS using the MRM conversions. To determine the stability analyte is kept at room temperature for 24 hours.

Construction of calibration curve

To obtain the linear response, five samples were taken on an alternate day by using similar plasma and stock solution. Each one of these contains nine concentration levels which differed from one another. These are named as inter and intra-day data results in stability and reproducible on alternate days. The peak area is calculated from extracted ion to the internal standard. The main purpose of an experiment is to assess the least level. Form the nominal concentration of the analyte calibration curve was constructed. Calibration curve of Tenofovir Disoproxil Fumarate is shown in Figure 3 and pioglitazone HCL is shown in Figure 4.

MRM transition optimization

To provide the spectra, MRM tracks the precursor ion and their multiple product ions that are quantifiable. They form different collision ions with each transition from parent ion to daughter ion. Each transition of collision energy is optimized by MRM transitions. Precursor ion is known as parent ion. Selective transmission of precursor ions (MS1) is passed through Q1 by m/z separation and are fragmented with collision energy to form product ions (MS2).

Product ions are known daughter ions. The m/z values of Tenofovir Disoproxil Fumarate are 520.13 →270.12 and Pioglitazone HCL is 357.07 →134.17. Table 2 shows the MRM transitions. Figure 5 & Figure 6 shows the product ion mass spectra of Tenofovir disoproxil fumarate and pioglitazone.

Linearity

It is defined as the relationship between analyte signals and analyte concentration in calibration samples. By using 1 $\times$ linear regression calibration curves are generated by using analyst 1.5 software. The linearity ranges differ from one ion source to others. In ESI source, the linearity is lost in high concentrations and at lower concentrations, linearity is maintained. Table 3 and Table 4 show the back-calculated standards.

Accuracy and precision

Inter and intra batch precision and accuracy values are shown below. Precision is expressed as a standard deviation. In a multistep preparation procedure, the results obtained show the acceptable accuracies. Accuracy refers to the closeness of true value and for measurement of uncertainty. The coefficients of variation are less than 15% for each analyte. The precision and accuracy analyzed by LQC, MQC and HQC are shown in Table 5. Table 6 shows the intra and inter run precision and accuracy. Figure 7, Figure 8, Figure 9, Figure 10 & Figure 11 shows the chromatogram of Tenofovir Disoproxil Fumarate and Figure 12, Figure 13, Figure 14, Figure 15 & Figure 16 shows the chromatogram of Pioglitazone HCL.

Recovery

The recovery is related to the separation approach inside variability stages. By comparing with the detector response, the recovery of an analyte is defined as the added amount of analyte to the extracted from the matrix. The average recovery rate of Tenofovir and pioglitazone is 93.9% and 99.85%. LQC, MQC and HQC values were also calculated as shown in Table 7 & Table 8.

Stability

Freezing and thawing quality control samples are validated by three cycles and the obtained values are shown in Table 9, which are within the range of inter-day precision and accuracy samples result in no instability. In autosampler, the quality control sample is kept for 24 hours at 4⁰C and the values obtained are within the range of inter-day QC samples leads to the deduction that no instability is found during the runtime. In Bench-top stability quality control sample are kept for 4 hours at room temperature and the values obtained are within the range of inter-day QC samples leads to the deduction that no instability is found during the runtime.

Conclusion

This method describes a simple, rapid, sensitive, specific, LC-ESI-MS/MS assay of Tenofovir and Pioglitazone for simultaneous detection and accurate measurement in human plasma using Metoprolol as an internal standard and to extract the analytes the precipitation technique was used. Linearity is fixed at 5 to 5000ng/mL. The m/z values of tenofovir were 520.13→270.12 and for pioglitazone 357.07→134.17. They calculated the Intra-day and inter-day precision and accuracy values at n=6. The average recovery rate of tenofovir and pioglitazone is 93.9%and 99.85%. No instability was found during the runtime.