Method development and validation of liquid chromatography-tandem/mass spectrometry for aldosterone in human plasma: Application to drug interaction study of atorvastatin and olmesartan combination.

In the present investigation, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of aldosterone (ALD) a hormone responsible for blood pressure in human plasma. The developed method was validated and extended for application on human subjects to study drug interaction of atorvastatin (ATSV) and olmesartan (OLM) on levels of ALD. The ALD in plasma was extracted by liquid-liquid extraction with 5 mL dichloromethane/ethyl ether (60/40% v/v). The chromatographic separation of ALD was carried on Xterra, RP-Column C18 (150 mm× 4.6 mm × 3.5 μm) at 30°C followed by four-step gradient program composed of methanol and water. Step 1 started with 35% methanol for first 1 min and changed linearly to 90% in 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 method was successfully applied for the drug interaction study of ATSV + OLM in combination against OLM treatment on blood pressure by quantifying changes in levels of ALD in hypertensive patients. The study revealed levels of ALD were significantly higher in ATSV + OLM treatment condition when compared to OLM as single treated condition. This reflects the reason of low effectiveness of ATSV + OLM in combination instead of synergistic activity.

INTRODUCTION

Primary aldosteronism (PAL) is a specifically treatable and potentially curable form of hypertension. The exact measurement of circulating aldosterone (ALD) is essential for the correct diagnosis of PAL. The importance of ALD measurement has greatly increased with the recent recognition that PAL is a more frequent cause of hypertension.[1] The current mainstay for measuring ALD is by antibody-based methods. These methods can be direct or require initial extraction of plasma or serum using liquid-liquid extraction, solid-phase extraction, or chromatographic methods.[23456] Overall, immunoassays have problems with varying selectivity and poor interlaboratory reproducibility, requiring each laboratory to establish its own reference range for the diagnosis of PAL.[7] For example, Schirpenbach et al.[8] recently reported a 2- to 3-fold difference in ALD concentrations measured by 4 currently used methods. Such discrepancies in ALD measurement between laboratories suggest a need for improved ALD measurement for both screening and confirmation of PAL.[910]

Gas chromatograph-mass spectrometer (GC-MS) has been used to measure ALD in biological fluids.[111213] Although this 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.

High-performance liquid chromatography (HPLC)-MS/MS is a powerful analytical technique that is, becoming increasingly used in the clinical setting.[14] HPLC-MS/MS offers the opportunity to provide more reliable measurement of ALD than immunoassays.[15] Of late, it has been shown that online solid-phase extraction coupled to HPLC-MS/MS was suited to plasma free metanephrine analysis and the diagnosis of pheochromocytoma.[16] Using this general approach, we report the development and validation of an HPLC-MS/MS method of measuring ALD that uses online semi-automated sample preparation. The given research study deals with the development and validation of a method for the determination of ALD in human plasma and apply the given method for drug interaction study in hypertensive patient.

MATERIALS AND METHODS

Chemicals and reagents

The standard powder of 5 mg ALD (A-9477) with internal standard (IS) ALD d-7 was procured from Sigma Aldrich. Dichloromethane, diethyl ether, methanol were of HPLC grade and were procured from Merck (Darmstadt, Germany). HPLC-grade organic solvents were purchased from Merck (Darmstadt, Germany). All chemicals were of analytical grade. Distilled water was prepared as required using an aqua MAX ultra, Younglin Instrument Co., Ltd. 60, Anyangcheondong-ro, Dongan-gu, Anyang-si, Gyeonggi-do, 431-836, The Republic of Korea, ultrawater purification system.

Instrumentation, chromatographic conditions and (MS)/MS condition

Instrumentation

The HPLC system consisted of Waters Alliance 2695 low pressure gradient separation module (Waters, Milford, MA) with the configuration of quaternary solvent delivery pump, sampler cooler, column heater. Mass specta data were acquired using a Micromass ZQ mass detector (Micromass, Ltd., United Kingdom).

Chromatographic conditions

The chromatographic separation of ALD was carried on Xterra, RP-Column C18 (150 × 4.6 × 3.5 μm) at 30°C. Optimized method was a four-step gradient program composed of methanol and water. Whole chromatographic separation was carried at flow rate of 0.25 ml/min, Step 1 started with 35% methanol for first 1 min and changed linearly to 90% in 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 sample volume was 20 μL as shown in Table 1.

Table 1

Gradient program of mobile phase for the determination of aldosterone by liquid chromatography-tandem mass spectrometry

Mass spectrometry/MS conditions

Mass spectrometry/MS detector was a Micromass ZQ mass detector (Micromass, Ltd., United Kingdom). The column was directly connected to the electrospray ionization probe operating at 350°C (disolvation temperature was 348-350°C and source temperature was 119-120°C). The sample temperature was 20°C with sample volume of 20 μL.

Preparation of calibration standards and quality control samples

The calibration standards were prepared by spiking human plasma with standard ALD with corresponding IS (ALD-d-7) to obtain resultant concentrations of 50, 100, 200, 400, and 800 ng/mL, respectively. The developed method was validated using three different quality control samples by spiking ALD in human plasma at 50, 200, and 800 ng/mL to represent low quality control (LQC), middle and high quality controls (HQC), respectively.

Sample extraction procedure

Sample extraction was done by liquid-liquid extraction using dichloromethane and ethyl ether in the ratio 60:40. For the same 1 mL of collected frozen plasma was added with 500 mL methanol/L of water was added before extraction. Thereafter, the extraction was carried out with 5 mL dichloromethane/ethyl ether (60/40) in a multi-tube vortexer. After centrifugation the upper organic phase was separated out in a conical flux and allowed to pass through nitrogen gas. Finally, the residue was dissolved in 350 μL of 350 mL/L methanol/water.

Method validation

Selectivity and specificity

Aldosterone is an endogenous compound present in humans, as a part of specificity studies plasma devoid of ALD was obtained from patients suffering from Addison's disease which are having no circulating ALD in them. The obtained plasma sample was processed by the liquid-liquid extraction procedure. The samples were chromatographed to determine to which extent endogenous plasma components may contribute to the peak interference at retention times of ALD. Furthermore, the eqi-volume of spiked plasma of standard ALD and aldo d-7 (IS) represent the exact location of peaks and ratio of extracted ALD from plasma with this methodology.

Linearity

Calibration curves were prepared by adding a known amount of ALD as well as aldo d-7, IS (50, 100, 200, 400, and 800 ng/mL) to 0.5 mL of blank plasma. The samples were extracted as described previously. The standard curves were constructed by plotting the peak area ratio of ALD Standard concentration and of ALD d-7 (IS) upon X- and Y-axis concentration ranges. Linearity was assessed by observing the values of coefficient of correlation (R2) of linearity plots, nearness of values near to one suggested linearity of responses. Furthermore, the linearity of each standard curve was confirmed by plotting the peak area of ALD. The unknown sample concentrations were calculated from the weighted (1/x2) linear regression analysis of the standard curve.

Precision and accuracy

Inter-and intra-day precision studies were done by injecting quality control dilutions (50, 200 and 800 ng/mL) as described earlier (n = 12) in developed chromatographic method. Peak areas were calculated for % relative standard deviation (RSD) values.

Extraction recovery

To calculate recovery of the extraction procedure, 12 replicates of quality control samples of ALD (50, 200, and 800 ng/mL) were extracted and analyzed. The peak area was compared with the same concentrations of unextracted standards of ALD reconstituted in methanol.

Stability

In bench-top stability, twelve replicates of low and HQCs of ALD (50 and 800 ng/mL) were analyzed at 0 and 6 h at room temperature and the deviation was calculated. In freeze-thaw stability, twelve replicates of LQC and HQC samples of ALD were prepared, frozen at -20°C and analyzed after two and three freeze-thaw cycles. Long-term stability was examined for 14 days by taking twelve replicates of HQC and LQC samples. The mean concentration was taken into consideration, which was compared with 0-day sample concentration.

Limits of detection and limits of quantification

The limits of detection (LOD) and limits of quantification (LOQ) were determined by injecting progressively low concentrations of the standard solution under the chromatographic conditions. LOD and LOQ were calculated directly from the calibration plot. LOD and LOQ were calculated as 3.3 σ/ S and 10 σ/S, respectively, where σ is the standard deviation of intercept and S is the slope of the calibration plot. The LOD was defined as a signal-to-noise (S/N) ratio of 3:1 and the LOQ was defined as an S/N ratio of 10.

Study design

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

The collection of blood plasma was basically done 3 times per individual for 7-14 days-in first stage without any drug administration. Second, along with combination of OLM + ATVS and third with only single hypertensive drug that is, OLM. First stage was for 1-week in which the patients were devoid of any antihypertensive drugs. The patients were evaluated for demography and measurement of BP followed by withdrawal of blood to quantify the levels of ALD.

RESULTS AND DISCUSSIONS

Selectivity and specificity (matrix effect)

Blank plasma obtained from patients suffering from Addison's disease was used for studies. The plasma was used to mark any endogenous interference. A representative chromatogram of the plasma blank is shown in Figure 1a. No additional peaks of endogenous substances were observed. Figure 1b shows the chromatograms of calibration standard containing ALD spiked in plasma with aldosterone d-7 (IS).

Figure 1a

Liquid chromatography-tandem mass spectrometry trace of without aldosterone spike to human plasma (quality control standard: 50 ng/mL)

Figure 1b

Liquid chromatography-tandem mass spectrometry trace of standard aldosterone (ALD) and ALD-d7 (internal standard) spiked in human plasma (blanked plasma sample)

Linear calibration curves Figure 2a with correlation coefficients near to 0.999399 were obtained over the concentration range of ALD (50, 100, 200, 400, and 800 ng/mL) to 0.5 mL of blank plasma. The coefficient of regression that is, r2 = 0.998 was obtained indicating linearity of results and an excellent correlation between peak area ratio for each concentration of ALD. The plasma spiked ALD standard versus ALD-d7 (IS) calibrated curve Figure 2b represents regression line linearity equation of y = 2.7917837018877 + 1.0014117703309×.

Figure 2a

Standard curve of calibrators' concentration of aldosterone

Figure 2b

Linearity curve plotting the peak area ratio of aldosterone (ALD)-d7 (internal standard) versus ALD (standard) concrete

Accuracy and precision

Method performance was evaluated as accuracy and precision as shown in Tables 2 and 3, determined by 12 replicate analyses for ALD at three concentration levels that is, LQC (50 ng/mL), MQC (200 ng/mL) and HQC (800 ng/mL), each on the same analytical run.

Table 2

Accuracy study of developed method for aldosterone in human plasma

Table 3

Precision study of developed method for aldosterone in human plasma (inter- and intra-day)

In bench-top stability, six replicates of LQC and HQCs of ALD (50 and 800 ng/mL) analyzed at 0 and 6 h at room temperature resulted in recovery in acceptable ranges, at 0 h the recovery of ALD from plasma was 96.5% for LQC and 103.1% for HQC, whereas after 6 h the extraction recovery of ALD was 93.4% for LQC and 100.2% for HQC. For LQC sample mean recovery for second and third freeze-thaw stability cycle were 98.3% and 96.8%, respectively, for HQC samples the mean recoveries for second and third freeze-thaw stability cycle were 105.2% and 102.8%, respectively, which were well inside the acceptable ranges as per the guidelines (i.e. ±15%). The method exhibited excellent sensitivity by demonstrating LOD of 0.13 ng/mL and LOQ of 0.432 ng/mL.

DISCUSSION

Inter-assay precision and accuracy were calculated after repeated analysis in three different analytical runs. Results concluded the repeatability of the method, including both sample processing and chromatographic measurement. Recovery results were subjected statistically analysis and %RSD were recorded. The %RSD is a ratio of standard deviation to mean in percent. %RSD values were small indicating good accuracy of results. Inter- and intra-day results also were good as the %RSD values were low [Table 4]. The recovery of ALD for freeze-thaw stability studies was found to be within the limits as per the guidelines. Long-term stability results for extraction recovery of LQC and HQC samples resulted in acceptable recoveries concluding the method suitable to stability studies for long periods. The method was confirmed for sensitivity by estimating LOD and LOQ. The method exhibited excellent sensitivity by demonstrating LOD of 0.13 ng/mL and LOQ of 0.432 ng/mL.

Table 4

The percentage extraction recovery of aldosterone from plasma

The developed liquid chromatography (LC)/MS/MS method for quantification of ALD in plasma was applied for the drug interaction study in hypertensive patients under the antihypertensive drug therapy. Blood was collected after dosing thereafter immediately the blood pressure was observed in lying posture for same individual patient in three stages and similarly with rest of the volunteer (patients). Table 5 shows that there were significant reductions (P < 0.001) in the blood pressure of volunteers with OLM when compared to without treatment stage.

Table 5

The effect of different antihypertensive treatment over the blood pressure (systolic/diastolic) of hypertensive human volunteers

The plasma concentration of ALD was calculated in the hypertensive human volunteers by using given developed method. There were significant decrease (P < 0.001) in the level of ALD in ATSV + OLM treated stage when compared to the without treatment stage in human volunteer. Whereas, the concentration of ALD in the OLM treated stage was lower than the detection limit as shown in Table 6.

Table 6

The effect of different antihypertensive treatment stage over the plasma concentration of aldosterone of hypertensive human volunteers

CONCLUSION

This study was undertaken to develop a sensitive method for quantification of ALD in human plasma. The developed LC/MS/MS method was validated in accordance to guidelines and confirmed to be specific, selective, linear, accurate, precise, and sensitive. The method exhibited acceptable recovery from spiked plasma samples also the method was proved to be sensitive demonstrating excellent LOD and LOQ levels of 0.13 ng/mL and 0.432 ng/mL, respectively.

The validated sensitive method was applied for pharmacokinetic studies to quantify the levels of ALD in human volunteers under therapy of ATSV, OLM, and combinations of both. The study concluded that the concentration of ALD in human volunteers treated with ATSV + OLM was higher than the OLM treated hypertensive human volunteer also the demographic characters revealed decrease in blood pressure of OLM treated patients when compared to ATSV + OLM treated ones. Thus, this study concludes failure of combination therapy for fulfilling the objective of synergistic activity in treating ALD mediated blood pressure as cardiovascular therapeutics, rather than the individual drug treatment [Figure 3].

Figure 3

Chromatograms of 12 blood plasma aldosterone trace in three different stages