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Year : 2015  |  Volume : 7  |  Issue : 1  |  Page : 49-55  

Assesment of endocrinal and biochemical entities through liquid chromatography-tandem mass spectrometry/mass spectrometer: Inter-relative investigation of the interaction based cardiovascular formulation

Department of Pharmaceutical Technology, Bioequivalence Study Center, Jadavpur University, Kolkata, West Bengal, India

Date of Submission23-Mar-2014
Date of Decision30-Jun-2014
Date of Acceptance02-Aug-2014
Date of Web Publication21-Jan-2015

Correspondence Address:
Tapan Kumar Pal
Department of Pharmaceutical Technology, Bioequivalence Study Center, Jadavpur University, Kolkata, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-7406.149813

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Background: Combinatory oral dosage treatment of atorvastatin (ATVS) and olmesartan (OLM) drugs to cardiovascular patients reflects unpredicted results instead of its individual therapy, which was accessed on quantification of endocrinal and biochemicals of plasma through liquid chromatography-tandem mass spectrometry/mass spectrometer (LCMS/MS). Objective: Mission was to track the remarkable biochemical variation in the plasma after induction of the combined formulation, to evaluate the pharma-market rumor on its efficiency. Methods: To fulfil undergoing research objectives for digging-up of market insult, human patient volunteers were chosen according to the required criteria along with bioethical regulation. A sensitive, rapid and precise method was developed and validated to estimate aldosterone (ALD), angiotensin (ANG-II) and the Mevalonate (MVA) not Mevalonic acid through LCMS/MS over least samples of cardiovascular patients. Level of each endogenous biochemicals were determined in three stages - without drugs, with a single drug (OLM/ATVS) and with their combination that was then correlate with blood pressure of respective volunteers. Result and Discussion: Comparative and correlative studies panaroma among these analytes was detected. The selectivity, specificity, linearity, precision, accuracy, extraction recovery, limit of detection and limit of quantification, stability were the essential points of validation of the developed methodology. And the significance of each endogenous analyte data were based on P ≥ 0.001. Thus, low value of ALD and reciprocally higher in ANG-II on administered single drug than its combination and equal concentration of mevalonate in both stages, was discovered. Conclusion: This concludes that the cardiovascular dosage formulation entrenched in the market are not synergistic and effective compared with a single drug as antihypertensive drug.

Keywords: Cardiovascular dosage, developed method and validation, drug interaction, endogenous biochemical, liquid chromatography-tandem mass spectrometry/mass spectrometer estimation

How to cite this article:
Das R, Pal TK. Assesment of endocrinal and biochemical entities through liquid chromatography-tandem mass spectrometry/mass spectrometer: Inter-relative investigation of the interaction based cardiovascular formulation. J Pharm Bioall Sci 2015;7:49-55

How to cite this URL:
Das R, Pal TK. Assesment of endocrinal and biochemical entities through liquid chromatography-tandem mass spectrometry/mass spectrometer: Inter-relative investigation of the interaction based cardiovascular formulation. J Pharm Bioall Sci [serial online] 2015 [cited 2022 Dec 7];7:49-55. Available from:

Although this gas chromatograms-mass spectrometer (GC-MS), analytical technique is considered a reference method that provides both accurate results and excellent specificity, these methods in general require extensive sample preparation including chemical derivatization. The lack of automation and complexity of sample preparation has relegated GC-MS to specialty clinical laboratories and is not used in routine clinical services. [1],[2],[3]

High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is a powerful, sensitive analytical technique that abandently used in the clinical trials. [4] HPLC-MS/MS offers excessive reliability measurement of aldosterone (ALD) than immunoassays. [5] Solid-phase extraction associate to HPLC-MS/MS was reliable to plasma free meta-nephrine analysis. [5] Thus, the development and validation of an HPLC-MS/MS method of determination of ALD in human plasma and its application for drug interaction study in the hypertensive patient.

It has been observed until now that all activity of peptide angiotensin II (ANG II) are associated with functions of type 1 (AT1) ANG receptor. AT2 have been proven to be a functional antagonist of AT1 receptor. [6],[7],[8],[9],[10],[11] There have numbers of reports of peptides with respect to the analysis of drugs in plasma using column switching techniques. [12],[13],[14] Analysis of the peptide has been performed using HPLC combined with radioimmunoassay. [15],[16],[17],[18] LC-MS is a useful tool for biological analysis and is widely used for identification and quantification of proteins and peptides. Usually, LC-MS is used to provide an initial identification based on molecular weight, LC-MS/MS provides further confirmation via structural specific fragmentation. The combination of LC-MS and LC-MS/MS analyses allows sensitive and unambiguous analysis of peptides in complex sample matrices. The main challenge in developing and validating a method for determining MVA in human plasma was that MVA is a polar, endogenous moiety that circulates in the blood stream at nanogram levels. In most methods, the extraction of MVA from plasma was carried out using ion exchange resins in the form of mevalonolactone. [19],[20] A precise, appropriate, and sensitive liquid chromatography-tandem mass spectrometry/MS (LCMS/MS) (negative-ion electrospray ionization) method was developed to evaluate mevalonic acid (MVA) in human plasma and also it was applicable to analysis MVA concentration in rat plasma after treated with rosuvastatin. [21]

   Methods Top

Market studies

The motive of research was developed from unpredicted result outcomes after investigation on patients getting chronic treatment on cardiovascular combined drug therapy and the best result in combination therapy with atorvastatin (ATVS) (anticholesteramics) rather than its individual hypotensive (olmesartan [OLM]). This combined regimen of specific drugs mentioned above was manufactured by a very famous pharmaceutical MNC in India.

Study design

The study on human volunteers was carried under approval of Ethics Committees "HURIP Independent Bioethics," Ibrahimpur Road, Kolkata, India. The study was performed along with patients' consent and under supervision of a doctor. Twelve hypertensive patients aged between 21 and 30 years, noninfected, not under any antibiotics, steroid and other medical therapy for a month, except specific cardiovascular drug provided by experts for this study. The patients were routinely supervised and also prescribed with a single category of medicines that is, OLM and ATVS, according to their therapeutic regimens.


Method developed and validation processes

method development and validation were carried over in thr three endogenous biochemicals analysis-ALD, ANG-II and the mevalonate (MVA). And their resultant data were interpreted to fix a conclusion.


The developed method was validated and was extended for application on human subjects to study drug interaction of ATSV and OLM on levels of ALD. The ALD in plasma was extracted by liquid-liquid extraction with 5 ml dichlormethane/ethylether (60/40% v/v). The chromatographic separation of ALD was carried on Xterra, RP-Column C18 (150 mm × 4.6 × 3.5 um) at 30°C, followed by 4 step gradient program composed of methanol and water. Step 1 started with 35% methanol for first 1 min and changed linearly to 90% in the next 1.5 min in step 2. Step 3 lasted for next 2 min with 90% methanol. The method finally concluded with step 4 to achieve initial concentration of methanol that is, 35%, thus contributing the total method run time of 17.5 min. The flow rate was 0.25 ml/min throughout the process. The developed method was validated for specificity, accuracy, precision, stability, linearity, sensitivity and recovery. The method was linear and found to be acceptable over the range of 50-800 ng/ml.


The ANG-II in plasma was extracted by with 5 ml methanol containing 5% formic acid through C 18 (Cartridges) liquid-liquid extraction, dried and reconstituted with reconstituted with 1 ml of 16% acetonitrile in 0.1% formic acid in water. The chromatographic separation of ANG-II with an Agilent Technology 6410 triple quadrupole (QQQ) was carried multiple reaction monitoring scan mode with an Agilent 1290 Infinity LC System for high-performance liquid chromatography. The sample was separated on a (Thermo Scientific) Hy-Purity advance (50 mm × 4.6 mm, 5 μm) using a mobile phase A - 16% acetonitrile in 0.1% formic Acid in water and mobile phase B - 0.1% formic acid in methanol at a flow rate of 0.3 ml/min, performed at ambient temperature. The mobile phase gradient of 16% acetonitrile in water was linearly increased to 38% acetonitrile over 10 min and subsequently the mobile-phase was increased to 100% acetonitrile over 15 min.


  • The method was validated and was impended for application on human subjects to study the concentration of mevalonate in plasma level after administration of ATVS individually and with a combination to OLM. The assay procedure involved the isolation of MVA from plasma samples using solid-phase extraction preconditioned cartridge, washed with methanol followed by 0.1 N HCI. The analytes were eluted with 3 × 0.5 ml of methanol and evaporated to dryness the Nitrogen stream. The residue was reconstitute for LCMS/MS analysis, were chromatographic separation was carried on a hypurity advance, 50 mm × 4.6 mm column with a mobile phase 10 mM ammonium formate (pH = 8) and acetonitrile. The flow rate was 0.8 ml/min throughout the process. The liquid chromatography, Agilent 1290 coupled to electrospray ion MS (MS QQQ). The developed method was validated for specificity, accuracy, precision, stability, linearity, sensitivity and recovery. The method was linear and found to be acceptable over the range of 50-1000 ng/ml.

Developed method and validation of endogenous biochemical analysis were utilized to evaluate the concentration levels in the human plasma, in three stages of patient volunteers - (1) Without drug, (2) With single (OLM/ATVS) drug (3) With a combined regimen (ATVS + OLM), in respect to a recovery area, reflecting the conc. and retention times of the traced mount of peaks. The comparative data interpretation on concentration. Level was carried-out within and between 20 patients. The study was structured to represents approaches of resultant variation, which draws an inter-relative matter between homogenous biochemical analysis and selected cardiovascular drugs formulation. Finally, the efficiency of individual drugs and its combination reflected.

   Results and Discussions Top

Method development and validation

A constitutional studies of data among all biochemical analysis behalf of linearity, accuracy and precision, extraction recovery, stability and sensitivity exhibits a best validator for endogenous biochemical quantity as well as qualities. The recovered area and retention time for specific concentration ranges was calibrated with standard versus internal standard chromatograms [Figure 1] to validated and trace/scan the chromatograms of plasma biochemical analytes of 12 patients.
Figure 1: Comparative chromatograms trace of standard and internal standard for concentration of aldosterone, angiotensin II and MVA at 50, 5, 50 ng/ml respectively under plotted recovered area versus retention time (minutes) with respect to multiple reaction monitoring scan

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  • Method performance was evaluated as accuracy and precision as shown in the [Table 1] and [Table 2], determined by 12 replicate analyses for all three bioanalytes at three concentration levels, that is, LQC (50), (5), (50) ng/mL, MQC (200), (50), (400) ng/mL and HQC (800), (200), (1000) ng/mL, each on the same analytical run. Inter-assay precision and accuracy were calculated after repeated analysis in three different analytical runs. The results concluded the repeatability of the method, including both sample processing and chromatography measurement. Recovery results were subjected statistical analysis, and percent RSD was recorded. The percent RSD is a ratio of standard deviation to mean in percent. Percent RSD values were small, indicating good accuracy of results. Inter-day and intra-day results also were good as the percent RSD values were low.
Table 1: Accuracy study of developed method for endogenous bioanalyte in Human plasma

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Table 2: Precision study of developed method for endogenous bioanalyte in Human Plasma (inter and intraday)

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Averaged for twelve measurements at each concentration level (n = 12).

% recovery = (response of extracted spike)/(response of postextracted spike) ×100.

Extraction recovery

recovery of MVA was determined by comparing peak areas obtained from extracted plasma samples with those found by extracting blank matrices through the extraction procedure and spiking with a known amount of MVA. The results showed that the mean extraction recovery of MVA was >85% at three different quality control like LQC, MQC, HQC respectively [Table 3].
Table 3: The percentage extraction recovery of endogenous bioanalytes from plasma

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In bench-top stability, 12 replicates of low and high-quality controls of ALD, ANG-II, MVA (50, 5, 50 and 800, 200, 1000 ng/mL respectively) analyzed at 0 and 6 h at room temperature resulted in the recovery in acceptable ranges, at 0 h the recovery of ALD, ANG-II, MVA from plasma was 96.5%, 95.6%, 98.4% for LQC and 103.1%, 101.2%, 101.7% for HQC, whereas after 6 h the extraction recovery of ALD, ANG-II, MVA was 93.4%, 92.5%, 95.3% for LQC and 100.2%, 98.2%, 100.9% for HQC respectively. The recovery of bioanalytes for freeze-thaw stability studies was found to be within the limits as per the guidelines. For LQC sample mean recovery for second and third freeze-thaw stability cycle were 98.3%, 96.9%, 97.1% and 96.8%, 94.8%, 94.5%, respectively, for HQC samples the mean recoveries for second and third freeze-thaw stability cycle were 105.2%, 102.3%, 103.4% and 102.8%, 101.6%, 1032% respectively, which were well inside the acceptable ranges as per the guidelines (i.e. ± 15%). Long term stability results for extraction recovery of LQC and HQC samples resulted in acceptable recoveries concluding the method suitable for stability studies for long periods.

Sensitivity studies (limit of detection and limit of quantification)

The method was confirmed for sensitivity by estimating limit of detection (LOD) and limit of quantification (LOQ). The method exhibited excellent sensitivity by demonstrating LOD of 0.13, 0.16, 0.97 ng/ml and LOQ of 0.432, 0.520, 2.33 ng/ml respectively.[22]

Evaluation of all thr three endogenous bioanalytes on chromatograms peak area in three stages, comparatively shows [Figure 2] the difference in concentration ratios of bioanalytes (ALD and ANG-II), where the ALD descends its order after evaluating on without the drug, with a combination (ATVS + OLM) and with single (OLM) administration. The status of ANG-II has been just reciprocal to ALD in all the three stages. In the case of MVA, conc. Level of without the drug is more than the rest of two stages-ATVS and combination, which were near about similar in concentration [Table 4]. The comparative study of concentrations of all the three types bioanalytes shows correlated effects among all 12 patient volunteers after examining without drugs, after administering with combination and single drug dosage [Table 4].
Figure 2: Comparative chromatograms of Biochemical analytes in three stages of a patients

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Table 4: The effect of different antihypertensive treatment over the blood pressure (systolic/diastolic) of Hypertensive Human volunteers

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The higher concentration of ALD in normal blood pressure (BP) patient collected at sharply 8:00 am, when the ALD is usually higher in plasma. However, after combination (ATVS + OLM) therapy, ALD seems to reduce in all twelve patients. On, individual therapy for OLM, response marked reduction in ALD level, which could be able to trace in LCMS/MS of this stated model. Similarly, it has been observed for ANG-II. In the case of MVA detection, the conc. of MVA is higher in normal BP patients and lower in with a single drug and its combination, such that it's apparently equal [Table 5].
Table 5: The effect of different antihypertensive treatment stage over the plasma concentration of endogenous bioanalytes of Hypertensive Human volunteers

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   Conclusion Top

After passing through all endogenous biochemical analytical investigations with a sophisticated LCMS/MS and after correlating all data from 12 patients on 3 stages of administrated therapy of cardiovascular dosage. It was concluded that, ATVS and OLM in combination shows lower effectiveness than its individual administration. Thus, the combined drug regimen yield drug interaction rather than expected synergistic activity.

   References Top

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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