New validated LC-MS/MS simultaneous estimation of Lopinavir and Ritonavir in human plasma positive ion mode using Deuterated internal standards

doi:10.21203/rs.3.rs-1430828/v1

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Research Article

New validated LC-MS/MS simultaneous estimation of Lopinavir and Ritonavir in human plasma positive ion mode using Deuterated internal standards

https://doi.org/10.21203/rs.3.rs-1430828/v1

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Aim

HIV protease inhibitors, used to treat AIDS patients, in order to run, it inhibits with the HIV protease enzyme during the final phases of viral replication. This was done by using high-performance fluid chromatography/mass spectrometry the researchers were able to determine three protease inhibitors specific, sensitive, and robust detection of lopinavir and ritonavir in human plasma.

Materials and Procedures

The excavation of analytes in solid matrix and their demineralized analogues serve as interior benchmark as a pre-treatment sample from 50 liters of human plasma. Chromatographic separation of mobile phase 5 mM methanol and ammonium acetate (85:15, v / v) and analytes conducted on a Hy-purity Advance 50 x 4.6 mm,5m ( Make-Thermo Scientific) column under gradient conditions.

Result

The technique was developed for lopinavir D8 concentrations of 20.00 g/ml and Ritonavir D8 at concentrations of 2,000 g / mL. The accuracy and precision of the analyzes, matrix effect, recovery and stability were evaluated according to US Food and Drug Administration standards.

Conclusion

Selectivity, Residual test, matrix influence, sensitivity uniformity and accuracy, stability, dilution integrity run size, and assessment of concurrent drug impact are all within acceptable parameters in this report. The simultaneous estimate of these inhibitors is possible due to the Sample preparation efficiency, fast analytical time and excellent selection. The technique has been verified and may be used to determine the plasma levels of These protease inhibitors are used in high-throughput clinical studies for aspect of the management.

The average lifespan of HIV-infected individuals has grown with the advent of protease inhibitors (PIs) for the therapy of the human immunodeficiency virus (HIV) in 1995, and their ongoing relevance in highly active antiretroviral therapy (HAART), lowering their death rate[1]. One or several regulators of nucleoside reverse transcriptase, as well as one or two PIs and one NNRTI, are used in the HAART regimen.This combination aids in the reduction of viral resistance and the prevention of negative outcomes [2]. HAVT-infected HIV-infected patients should receive RTV-enhanced protein inhibitors such as other antiretroviral drugs include darunavir and atazanavir as part of their first treatment [3].The use of PIs for HIV infection therapy has risen due to the fast development of resistance to NNRTIs. PIs primarily affect the aspartate protein enzyme of the human immunodeficiency virus (HIV PR), which breaks down viral gag and gag-pole protein intermediates into fully functioning viral enzymes and matrix proteins [4]. Abbott Laboratories developed the most powerful first-generation PI, RTV, the FDA gave its approval in 1996. It is now utilised as a booster to enhance the pharmacokinetics of other PIs since it exhibits anti-HIV-1 and anti-HIV-2 proteolytic action. Its therapeutic benefits. Norvir [5] is a brand name. ATV and DRV are second-generation protein inhibitors used to inhibit protein strains that are resistant to first-generation inhibitors. Additionally, both medicines are well tolerated and have little adverse effects. Ciba-Geigy developed ATV, an azapeptide, which Bristol-Myers Squibb marketed as Reyataz. It was licensed developed 2003, and it has a distinct HIV resistant characteristic as well as pharmacokinetics that enable for a single daily dosage [6]. It is unique against mutation and high resistance proteins because of its capacity to adapt to the putative ‘substrate envelope' of the active site [1]. DRV is 100 actually more powerful against wild-type HIV-1 proteases than several other PIs.Pharmacokinetic measures, dose optimization, and drug-drug interaction investigations all need accurate estimation of PI plasma concentration. Despite the fact that these PIs offer a wide range of clinical benefits, their therapeutic index is extremely limited, necessitating therapeutic drug monitoring [8]. Methods such as ELISA, HPLC-UV, LCMS/MS, and UPLC-MS/MS were used to determine RTV [9, 10] as a single analyte. Checa et al. [2] evaluated antiretroviral drug detection techniques up to 2008, Focusing on key analytical methodologies for conducting clinical samples. Since then, many additional techniques have been published that report simultaneous detection of these medications in human plasma [10–16], dried blood smears and humans’ entire blood [17, 18], Mononuclear cells in the peripheral blood [19, 20], rat serum and tissue [21] in different matrices. Bioanalytic for antiretroviral for drugs in various tissues and body fluids such as prenatal fluid, cervical fluid, in a recent excellent review study, cerebrospinal fluid, extracellular cerebrospinal fluid, saliva, and male seminal plasma / serum were characterized [22]. The majority of these techniques used the LC-MS/MS technique for simultaneous ARV analysis [10, 12–15, 17–20], with just a few publications [11, 16, 21] mentioning UPLC-MS/MS technique is being used for research. Yadav et al. [11] used UPLC-ESI-MS/MS to evaluate lopinavir (LPV) and RTV in plasma samples in order to conduct a bioequivalence study in human healthy volunteers. Another study used UPLC-MS/MS to identify Indinavir (IDV), LPV, and RTV are three protease inhibitors. Huang et al. [21] are a group of researchers that have worked on a number of different projects RTV, IDV, ATV, and Efavirenz nanoparticle concentrations in blood and rat tissues were measured using UPLC-MS / MS. However, no UPLC-MS/MS technique exists to concurrently detect ATV, DRV, and RTV in human plasma. By expanding the popular concepts of liquid chromatography, (UPLC) has added a new level to the science of segregation.To improve resolution, sensitivity, and throughput, it uses sub-2 particle sizes. Managing the volume of the system and the peak divergence allows UPLC to decrease analysis time and enhance chromatographic performance over HPLC. When compared to traditional 4.6mm id columns, solvent usage may be reduced as well [23]. Thus, a robust, selective, and fast ATV, DRV, and RTV in human plasma may be accurately detected using the UPLC-MS/MS method has been established and thoroughly validated in the current study. For sample preparation, the technique uses just 250L plasma volume and has high effectiveness of chromatography (2.5 min). It may be utilised in an elevated clinical setting as well as for medication monitoring.

2.1Chemicals substances are number two on the list of things to be aware of Clearsynth Labs Pvt. Ltd. provided lopinavir D8 (98.05%) and ritonavir D8 (95.15%) reference standards, as well as their deuterated internal standards (ISs) (Mumbai, India). Mallinckrodt Baker, S.A.de C.V. provided us with HPLC-grade methanol and acetonitrile (Estado de Mexico, Mexico). Merck supplied the bio ultra-grade ammonium acetate and the LC-MS grade formic acid (St. Louis, MO,USA). BOND ELUT PLEXA from Agilent Technologies supplied Oasis HLB (1 cc, 30mg) extraction cartridges. The Milli-Q water purifying equipment from Millipore In-house was used to prepare the water for the research. Supratech Micropath (Ahmedabad, India) provided blank human plasma in K2EDTA, which was kept frozen until needed.

2.2. Mass Spectrometry and Liquid Chromatography Conditions

The Waters Aquity UPLC System (MA, USA) with an increased advances was used to undertake chromatographic analysis of LOP and RTV (50 4.6 mm, 5 m) 35C analytical column. The phase of (a) methanol and (b) 5 mM ammonium acetate (85:15, volume / volume) was separated using a gradient program with 0.800 mL / min and 50% flow splitting.The A:B ratio was maintained at 50:50 (v/v) for the first 0.8 minutes, then adjusted to 30:70 (v/v) for the next 0.8 minutes. Up to 2.0 minutes, the system was equilibrated to its original conditions. The sample manager was kept at 10 degrees Celsius with a 3-degree Celsius warning band, and the system's average pressure was 6000 pounds per square inch. The Quattro Premier XE Mass Spectrometer is used to detect and calculate (MRM) analyzes and ISs in the positive electrospray ionization mode of API-3000 LC-MS / MS.Table 1 summarises the optimum mass parameters for analytes and ISs that are source and compound dependent, as well as MRM transitions. All UPLC and MS settings are controlled by MassLynx version 4.1.

2.3 Preparations of Quality Control Samples, Calibration Standards, and Standard Stock

The standard solutions of Lopinavir and Ritonavir was accurately weighed at 2,000 mg workplace conditions, then transferred to a clean 2 mL glass conical flask and diluted in methanol, and obtained a volume composition of 1 mg / mL at the same volume.Corrected the concentration of Lopinavir and Ritonavir solution above, taking into consideration its potency and the real weight. The stock solution was kept in the refrigerator for up to 6 days at 2–8°C. Both Lopinavir and Ritonavir stock solutions were made independently.

For plasma infusion to produce Standard solution are diluted to suitable levels using a combination of methanol and milliQ water (dilution) in a ratio of 20:80 v. / V) for calibration curve parameters (CC), quality control (QC) samples, and DIQC samples. In the mobile phase, all additional final dilutions are made (system fit dilution, aqueous mixture, etc.).

2.4 Internal Standards for Lopinavir D8 and Ritonavir D8 Stock Solution:

About 2.000 mg of Lopinavir D8 and Ritonavir D8 working standards were carefully weighed and they are diluted in HPLC grade methanol in a clean 2 mL glass volumetric flask, and the quantity is likewise adjusted to generate a 1 mg / mL solution.Modified the aforementioned concentrations of Lopinavir D8 and Ritonavir D8 solution, taking into account their potency and the real weight. The stock solution was kept in the refrigerator for up to 6 days at 2 – 8 °C. Lopinavir D8 and Ritonavir D8 stock solutions were produced separately. For internal standard dilution, the stock solutions were diluted to an appropriate concentration using diluent.

2.5 Sample Extraction Protocols

Solid-Phase Extraction

SPE is the separation of a liquid (a sample matrix or solvent containing an analyzer) from a solid (sorbent). This sample treatment technique allows for the concentration and purifying of solvents from solvent by solvation on consists, as well as the purifying of extract following extraction. The selective adsorption (absorption on the surface) process underpins SPE.

Step 1: Conditioning

Prior to sample application, all SPE tubes must be conditioned with the necessary solvents. Conditioning is required in order to:

Activate the stationary phase bed's sites.
Avoid drying the sites and maintain them in the arising posture.
Remove any dust, moisture, or other pollutants from the process.
Before applying the sample, remove any surplus conditioning solvent.
As conditioning solvents, MeOH, tiny quantities of DCM, t-BME or another organic solvent, water, and buffer are frequently employed.

Step 2: Pre-treatment of the samples:

This entails shifting the intended sample size to the sample tube specified for itor adding the required internal standard volume to the sample, rotating the sample, or adding an acceptable buffer adding chemical to the material, and then spinning it.

Step 3: Samples Application:

Apply using a moderate flow rate without separating the sample from the top of the cartridge, taking into account that there is no pattern drop on the inner wall of the cartridge; Analyzer needs a slow flow rate to interact with adsorbent‌ and analyzer retention can be achieved due to connection that is just momentary.

Step 4: Rinsing

It is intended to eliminate matrix components or other disruptions from the cartridge by rinsing it with a poor dilute solution or a mixture of solvents or solution, and it is applied to reduce poorly retained matrix components or other interferences from the cartridge by washing it with a weak dilute solution or a solvent mixture or solution interventions are removed from the cartridge prior to analysis.Water and pH buffers are used as rinsing or washing solvents.

Step 5: Allow to dry

With the assistance of a vacuum pump, provide adequate suction for the required time duration; the suggested drying time is 2-3 minutes. It's designed to get rid of any leftover washing solvents or buffers. Avoid any potential for contact or precipitation. Eliminate the possibility of cartridge obstruction owing to the production of air bubbles during the elution process.

Step 6:Elution

Purification is the process of sending a powerful Slowly pumping solvent and through cartridge allows it to soak further in the packages for optimal extraction effectiveness. Methanol, ACN, acidic or alkaline methanol, ACN, tiny quantities of DCM, T-BME, and any other solvent mixture with methanol, ACN, or any other appropriate chemical are used as emulsion solvents.

2.6 Matrix Biological

For the selectivity assay, twelve batches of human K2-EDTA plasma were tested, including one lipid plasma and one hemolytic plasma. For lopinavir and ritonavir, a total of 12 groups of human plasma, including hemolytic and blood plasma, were determined to be without any internal effect, in addition to the internal criteria. All calibration parameters, quality control models and DIQC models were demonstrated from the six plasma batches collected.

After high growth, Alcots of 300 μl of indented plasma extract was taken into prepared microcentrifuge tubes for CCs and 400 μl for QCs, and all bulk serrated samples were kept in the cold room at 70 ° C with the exception of twelve duplicate LQC. HQC is kept in a cold room at -20 ° Cto generate stability data.

2.7 Quality Control Samples and Calibration Curve Standards

A standard titration curve has been established, with nine lobinavir concentrations range from 50.587 ng/mL to 7026.018 ng/mL. and ritonavir 5.075 ng / mL to 704.840 ng / mL.Lopinavir concentrations were 50.866 ng/ml (LLOQ QC), 142.084 ng/ml (LQC), 710.422 ng/ml (MQC1), 3229.190 ng/ml (MQC2), and 6047.172 ng/ml (HQC), and Ritonavir concentrations were 5.092 ng/ml (LLOQ QC), 14.223 ng/ml (LQC), 71 Until they were used, these samples were kept at –70°C.

3.1.5 mM acetate of ammonium

A 1000 mL reagent bottle containing 1,000 mM Q of water was filled with about 385.4 gramme of ammonium acetate. In an ultrasonicator, fully combine the ingredients and sonicate for 5 minutes. After preparation, the buffer solution was stored at room temperature (20 5 C) and utilised within four days.

3.2 Phase of Mobility (85:15, v/v)

In a 2000 mL reagent container, 300 mL of 5 mM ammonium acetate solution was then transferred, and 1700 mL of HPLC grade methanol was injected. They were thoroughly mixed and sonicated for 5 min on an ultrasound machine. The aqueous layer was stored at room temperature (20 5 C) and utilised within 7 days of being prepared.

3.3 Dilutant (v/v)

Diluted, A 20:80 volume ratio of HPLC grade methanol and milli cue water was created. It was then sonicated for 5 min on an ultrasound machine. The diluent was kept at room temperature (205°C) and used within seven days after preparation.

3.4 Rinsing solution (v/v)

The rinsing solution was diluent.

(w/v) 100mM ammonium acetate buffer:

A 1000 mL reagent vial containing 1000 mL Milli Q water was filled with 7.70 g of ammonium acetate. Mix thoroughly and sonicate for 5 minutes in an ultra-so nicator. After synthesis, the blank solution was stored at room temperature (20 5 °C) and utilised within four days.

3.5 Two percent formic acid buffer:

Two mL formic acid was added to 98 mL Milli Q water in a 100 mL reagent container. Mix thoroughly and sonicate for 5 minutes in an ultrasonicator. The solution was produced as needed and utilised within four days after its production. The buffer solution was kept at room temperature (20 ± 5 °C).

Table 1: Optimization data

Parameter	Lopinavir	Lopinavir D8	Ritonavir	Ritonavir D8
Ionization mode	+ve	+ve	+ve	+ve
Detection m/z	629.30-parent & 447.30 -product	637.40-parent & 447.30-product	721.40-parent& 296.20-product	729.60-parent & 304.30-product
Ion Spray Voltage (IS)	5000 V	5000 V	5000 V	5000 V
Temperature (TEM⁰C)	500	500	500	500
Curtain gas (CUR)	8-psi	8-psi	8-psi	8-psi
Collision gas (CAD)	8-psi	8-psi	8-psi	8-psi
Nebulizer Gas (NEB)	9-psi	9-psi	9-psi	9- psi
Declustering Potential (DP)	46V	46V	46V	46V
Focusing Potential (FP)	170V	170V	170V	170V
Collision Energy (CE)	21V	30V	26V	30V
Collision Cell Exit Potential (CXP)	12.00 V	12.00 V	8.00 V	12.00 V
Entrance Potential (EP)	10V	10V	10V	10V
Dwell time (msec)	200.0	200.0	200.0	200.0

Column: Hy PURITY ADVANCE 50*4.6 mm, 5µm (Make: Thermo Scientific)
5 mM Ammonium Acetate (85:15, v/v) Mobile Phase: HPLC-Grade Methanol
HPLC Grade Methanol: Milli Q / HPLC Grade Water (20:80, v/v) Rinse Solution
Rate of flow: 0.800 ml per minute (with divider)
50:50 splitting ratio of lits
Taster coolant temperature: 10°C
Temperature Oven Column: N / AP
Amount of injection: 15μl
Amount of needle rinse: 1000 μl
Modes of purifying: both during suction
Holding time: Lopinavir 1.00 ± 0.3 min
Lopinavir D8 1.00 ± 0.3 minutes
Ritonavir 1.00 ± 0.3 minutes
Ritonavir D8 1.00 ± 0.3 minutes
Run Time:2.50 minutes

To ensure a complete mix of contents, The samples were placed and vortexed at room temperature. RIA was used to add the 250-l plasma samplelead tubes, co-diluted to 25 µl internal standard (20,000 µg / mL Lopinavir D8 and 2,000 µg / mL Ritonavir D8) and, with the exception of empty plasma samples, obtained by 25 µl dilution and vortexing. A 200µl formic acid buffer solution (2% formic acid) was added and vortexed.

The sample set was placed in pre-conditioned Bond Elute Plexa pellets (30 mg / mL) a buffer solution made up of 1.0 mL HPLC methanol, 1.0 mL Q water, and 1.0 mL formic acid (2%) (new cartridge). Each (as an example). The extraction capsules was filled with 1.0 mL of ammonium acetate solution in a 100 mL bottle. The sample was filtered in a 1,000µl mobile phase before being injected into the LC-MS / MS device.

Optimization of data is represenred in Table No.1.Representatives for aqueous standard analysis and internal standard mixture, plasma vacuum, plasma vacuum according to the standard solution MQC1 sample, MQC2 sample, HQC sample, LLOQ QC sample, regression analysis and analyzed blank plasma sample for Lopinavir analysis. Calibration curves for Lopinavir and Ritonavir are shown in Figure 1 & 2.Calibration curve results are shown in table 2.

Shapes:

CC: Calibration Curve
Lower Limit of Quantification Quality Control (LLOQ QC) is a term used to describe the lower limit of quantification.
LLOQ stands for Lower Limit of Quantification.
LQC stands for low quality control.
MQC stands for Medium Quality Control.
HQC stands for "high quality control."
DIQC: DIQC (Dilution Integrity Quality Control) is an acronym for Dilution Integrity Quality Control.

A typical chromatogram for blank sample, Aqueous and Internal Standard mixture and Blank Plasma Chromatogram with Internal Standard Samples of Lopinavir and Ritonavir are shown in figure 3,4 and 6 respectively.

Representative Chromatogram of LOQ sample without Internal standard, LLOQ,MQC and HQC are shown in Fig.5,7,8 and 9 respectively.

6.1 Pre-Validation

6.1.1 Carryover test is a test that is used to determine whether or not something has been

6.1.2 Selectivity,

6.1.3 Sensitivity, and

6.1.4 Matrix Effect

6.2 Validation

6.2.1 Linearity

6.2.2 Accuracy and precision

6.2.3 Recovery

6.2.4 Integrity of the dilution

6.2.5 Ruggedness

6.3 Affirmations of stability

6.3.1Stability of the temperature in the room

6.3.2 The stability of refrigerator stock solutions

6.3.3 Stability of the benchtop

6.3.4 Stability of the auto sampler

6.3.5 Stability over the short term

6.3.6Stability of the freeze-thaw cycle.

6.3.7 Extract Stability in Wet Conditions

6.3.8 Stability during re-injection

6.3.9 Adverse drug interactions

6.3.10 Evaluation of the run size

6.3.11 Stability in Whole blood.

Selectivity:

Observation of the mass transitions of Lopinavir, Ritonavir, and internal standard on Representative chromatograms of empty plasma collected from plasma batches do not show significant overlap from internal components.

Acceptance Criteria :

The internal standard should be £ 5% of the average internal standard response in LLOQ samples, whereas the maximum levels of multidisciplinary interference during retention should be £ 20% of the average reaction response in LLOQ samples.

After failing to meet one or more of the above acceptance criteria, try again sooner or later. Although the experiment was repeated, it did not meet the acceptance criteria; Therefore, the method should be changed and documented in accordance with the standards.

Table 2: Data for standard curve

SEL-LLOQ	Analyte's Catchment Area	Internal Standard Area
1.	82992	660702
2.	84950	638557
3.	75903	626177
4.	79760	632615
5.	83090	611521
6.	80361	681546
Mean	81176.0	641853.0
SD	3214.91	25269.71
% CV	3.96	3.94

Sensitivity:

Lopinavir and Ritonavir's LLOQ concentrations in human plasma were set at 50.587 ng/mL and 5.075 ng / ml, correspondingly. The accuracy and precision of Lopinavir were found to be 2.73 percent and 96.67 percent, correspondingly. The precision and accuracy of retonavir were also found to be 5.99 percent and 97.13 percent, respectively.

LLOQ models must have an accuracy of 20% of the nominal value and 20% accuracy. Injected LLOQ samples should be within 20% of the nominal value in at least 67% of cases (4 out of 6).

Linearity:

In plasma samples, the regression equation for lopinavir and ritonavir is best calculated using a weighting of 1 / (concentration ratio) 2 of concentration to IS. Concentration range from 50.587 ng / ml to 7026,018 ng / ml is higher than r 0.99 for Lopinavir and Ritonavir.

Acceptance Criteria:

≥ 0.98 r² (r=coefficient of correlation)
Nominal LLOQ concentration ± 20 % off.
Variation of ±15% from the nominal voltage of the criteria rather than the LLOQ concentration
At least 75% of the standard calibration standards, including the lower limit of detection (LLOQ) and the maximum concentration, must be present, meets the above criteria.

Selection, relay and matrix effects, sensitivity, simplicity, precision and accuracy; More; Samples were diluted safety run size assessment; recovery; and concomitant drug effect tests are all within the acceptable range for Bioanalytical method, according to this report. All results of validation are represented in Table 3. Different stability analysis is done and results are shown in Table 4.

Table 3: Data for validation parameters

Analyte		% Nominal / %Stability	Precision	% Nominal / % Stability	Precision
Matrix		Plasma	N/AP	Plasma	N/AP
Detection		m/z 721.40 (parent) and 296.20 (product)		m/z – 729.60 (parent) and 304.30 (product)
Range		5.075 ng/mL – 704.840 ng/mL		N/AP	N/AP
Minimum Quantifiable Concentration		5.075 ng/mL		N/AP	N/AP
Matrix Effect	IS Normalized Matrix Factor at LQC	0.983		N/AP	N/AP
	IS Normalized Matrix Factor at HQC	0.984		N/AP	N/AP
Sensitivity		97.13%	5.99%	N/AP	N/AP
Coefficient of correlation (r²)		0.9978- 0.9996		N/AP	N/AP
Within Batch Precision and Accuracy		98.09%-110.85% (LLOQ QC) 101.39%-105.27% (LQC), 97.35%-99.21% (MQC1) 98.07%-100.48% (MQC2) 100.74%-101.74% (HQC)	2.73%-8.77% (LLOQ QC) 1.23%-4.64% (LQC) 1.53%-3.49% (MQC1) 0.69%-2.00% (MQC2) 1.02%-2.66% HQC)	N/AP	N/AP
Intra Day Precision and Accuracy		104.74% (LLOQ QC) 102.99% (LQC) 98.49% (MQC1) 100.28% (MQC2) 100.80% (HQC)	3.08% (LLOQ QC) 3.11% (LQC) 1.85% (MQC1) 1.57% (MQC2) 1.92% (HQC)	N/AP	N/AP
Within Batch Precision and Accuracy for Stabilities (Freshly Spiked QC’s)		105.76% (LQC) 108.06% (MQC1) 103.07% (MQC2) 103.82%(HQC)	3.10% (LQC) 2.26% (MQC1) 4.99% (MQC2) 2.45% (HQC)	N/AP	N/AP

Table 4: Stability data

Wet extract stability (44 hrs)	103.92%-106.73%	1.88%-2.15%	N/AP	N/AP
Freeze Thaw Stability (V Cycles)	103.34%-105.25%	0.86%-2.54%	N/AP	N/AP
Bench Top Stability (8 hours)	104.77%-106.06%	2.58%-3.44%	N/AP	N/AP
Short term -20°C Stability (5 days)	101.20%-101.34%	2.52%-3.57%	N/AP	N/AP
Recovery	90.57%	1.08% -2.57%	91.74%	3.27% -6.40%
Dilution Integrity: Two times dilution	103.30%	1.89%	N/AP	N/AP
Dilution Integrity: Four times dilution	97.81%	2.81%	N/AP	N/AP
Within Batch Precision and Accuracy (Ruggedness)	110.85%LLOQ QC) 105.27%(LQC) 99.17%(MQC1) 99.31% (MQC2) 101.69% (HQC)	8.77%(LLOQ QC) 4.64%(LQC) 3.49%(MQC1) 0.97%(MQC2) 1.29%(HQC)	N/AP	N/AP
Re Injection Stability (51 hrs)	97.68%-97.84%	2.18%-3.72%	N/AP	N/AP
Room Temperature Stock Solution Stability (17 hrs.)	102.14%	1.07%-1.79%	99.99%	1.98%-2.25%
Room Temperature Spiking Solution Stability (17 hrs.)	102.31%	1.07%-1.32%	101.78%	1.66%-2.25%
Frozen Stock Solution Solidity (6 days)	99.65%	1.42% -1.94%	100.49%	1.36% -2.43%

HPLC-MS / MS

High performance liquid chromatography tandem mass spectroscopy

LOQ

Limit of Quantification

LQC

Low quality control samples

MQC

middle quality control samples

HQC

High quality control samples

It has been shown that the analytical method described above can be successfully used to estimate Lopinaviral and Ritonaviral concentrations Lopinavir m/z 629.30 (parent) and 447.30 (product), Ritonavir m/z 721.40 (parent) and 296.20 (product), Lopinavir D8 m/z – 637.40 (parent) and 447.30 (product) and Ritonavir D8 m/z – 729.60 (parent) and 304.30 (product) were detected in human plasma over the range of 50.587 The HPLC-MS / MS technique for the detection of Lopinavir and Ritonavir in human plasma fulfilled the approval requirements.

Acknowledgements: The authors express their gratitude to Anacipher lab,Hyderabad for allowing to conduct all experimental works in their lab.

Author contributions:

Dr.Nalini Kanta Sahoo: Conceptualization, Methodology, Experimental, Writing- Original draft preparation, Project administration.

Bui Thanh Hung, and Prasun Chakrabarti:supervision of whole of the research work and analysis of the data obtained.

Shweta Singh Chauhan: Conceptualization, Writing- Reviewing and editing.

P.Chaitanya kiran: Resources –Experimental work, Conceptualization, Data curation.

Madhusmita Sahu: Formal analysis, Application of statistics.

Funding: Not applicable.

Availability of data and materials:

All data generated or analysed during this study are included in this published article.

Ethics approval and consent to participate: Collborative work with Anacipher lab (Ethically approved).

Consent for publication Not applicable.

Competing interests

All the listed authors have read and approved the submitted manuscript. This manuscript and data, or parts thereof, have not been submitted for possible publication to another journal, nor has the work been previously published elsewhere

The authors declare that they have no competing interests.

Author details

¹SRM Modinagar College of Pharmacy, SRMIST (Deemed to be University), Delhi-NCR Campus,Modinagar,Ghaziabad, Uttar Pradesh, 201204

²Director of Artificial Intelligence Laboratory, Faculty of Information Technology, Ton Duc Thang University,19 Nguyen Huu Tho street, Tan Phong ward, District 7, Ho Chi Minh City, Vietnam.

³ITM SLS Baroda University, Dhanora Tank Road, Paldi Village, Halol Highway, Near Jarod,
Vadodara - 391510 (Gujarat), INDIA.

⁴School of Pharmacy, Arka jain University, Gamharia, Jharkhand, 832108

⁵Yalamarty Pharmacy College, Tarluwada, Anandapuram, Visakhapatnam,A.P, 530052

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New validated LC-MS/MS simultaneous estimation of Lopinavir and Ritonavir in human plasma positive ion mode using Deuterated internal standards

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Abstract

Figures

1. Introduction

2. Methods And Materials

3. Solution

4. Sample Preparation

5. Results

6. Validation Parameters

Abbreviations

Conclusion

Declarations

References

Competing Interests

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