INTRODUCTION:

Moxonidine is new-generation centrally acting antihypertensive drug for the treatment of mild to moderate essential hypertension. It may have role when thiazides, beta-blockers, ACE inhibitors and calcium channel blockers are not appropriate or have failed to control blood pressure. It demonstrates favorable effects on parameters of the insulin resistance syndrome, apparently independent of blood pressure reduction. Moxonidine is selective agonist at the imidazoline receptor subtype 1. This receptor subtype is found in both the rostral ventro-lateral pressor and ventromedial depressor areas of the medulla oblongata. Moxonidine therefore causes decrease in sympathetic nervous system activity and, therefore, a decrease in blood pressure. Compared to older central-acting antihypertensives, moxonidine binds with much greater affinity to imidazoline I₁-receptor than to the α₂-receptor¹.

Moxonidine molecular formula is C₉H₁₂ClN₅O with molecular weight is 241.677 g/mol. The bioequivalence studies under fasting conditions are in accordance with CPMP/EWP/QWP/1401/98 Note for Guidance on the investigation of bioavailability and bioequivalence².

Moxonidine is rapidly absorbed after oral administration. In humans, 90% of oral dose is absorbed. Ingestion of food has no effect on the pharmacokinetics of Moxonidine. There is no first-pass metabolism and bioavailability is around 88%. About 7% of moxonidine is bound to human plasma proteins (Vdss = 1.8 ± 0.4 l/kg). Peak plasma levels of moxonidine are reached 30-180 minutes after administration of film-coated tablet. The mean plasma elimination half life is 2.2-2.3 hours and the renal half-life 2.6-2.8 hours³.

As per OGD, Single dose Bioequivalence fasting study to be conducted for new generic formulations in healthy population to enter into the USA market.

Highly sensitive (5pg/ml LOQ) and selective, rapid (3 min), MRM method has been developed and validated for the estimation of Moxonidine in human plasma (K₂EDTA as an anticoagulant) by simple isocretic liquid chromatography with 10 microliter injection on tandam mass spectrometry system, method involves sample processing on solid phase extraction with very low matrix effects and highly selective and rapid, Clonidine used as internal standard. The method has successfully validated and applied to analyze bioequivalence study samples of ~360.

Moxonidine is a low molecular weight basic polar drug. Basic functional groups are active for extraction and ionization. Because of the drug polar in nature, we got matrix effect problems but clonidine (ISTD) is compensating those, IS normalized matrix factor is within limits. Recovery found ~40% due to matrix effects but recovery is consistent and reproducible. Selectivity, Precision and accuracy found acceptable.

No method is published on public domain for the estimation of Moxonidine in plasma by LC-MS/MS with 5pg/ml and 3 minutes run time. Minxia M. He and others, have reported a method with high duration gradient LC program for 27 minutes for 0.05 to 8 ng/ml CC range with Solid phase extraction method using unusual Trifluoroacetic acid in elution solvent on mass spectrometer operated with APCI and the sample was concentrated to 150micro liters and 125 micro liters injection volume⁴, this method involves high duration with many complex steps like gradient LC, trifluroacetic acid, APCI, 125 microliter injection. Stephen D Wise and others published a method which involves APCI with 0.25 ng/ml LOQ⁵, Luhia zhao and others have recently reported a LC-MS⁶, SIM methods are not sensitive and selective, LC-MS/MS methods got more advantages over SIM methods, Other methods reported are to estimate moxonidine in tablet dosage form which do not have direct comparison with the current method^7-15.

MATERIALS AND METHODS:

Materials:

Moxonidine and Clonidine (ISTD) standards are received from VLS, Mumbai, India, Chemicals purchased from JT Baker Inc. USA. Positive pressure solid phase extraction manifold of Orochem, USA was utilized for processing. Bond elute Plexa (1CC 30mg) cartridges procured from Agilent technologies, USA whereas blank plasma received form the inhouse clinical department. Study protocol was reviewed and approved by Aavishkar Ethics Committee, Goa, India.

Methods:

1mg/ml stock solutions of Moxonidine and Clonidine were prepared in methanol, further stock dilutions and spiking solutions are prepared in 50% methanol in water solution, then stored in refrigerator. 5% spiking solutions were spiked in blank plasma to archive required CC and QC sample concentration. 8 point calibration curve over the range 5 to 10000pg/ml is selected whereas 4 Quality control levels are incorporated within the CC range. Spiked samples are stored at -70⁰C until analysis.

Sample Extraction Procedure:

Add 50µl of IS Dilution to samples except Blank sample where add 50µl of diluent, Aliquot 0.500ml of sample and vortex, Add 0.500 ml of Water and vortex, Condition the SPE cartridges with 1.000ml methanol followed by 1.000ml water, Load the prepared sample in the SPE cartridges, Wash cartridges twice with 1.000ml of water followed by twice with 1.000ml of 5.00% methanol in water and dry the cartridges for ~2 min. Elute the samples with 0.200ml of the mobile phase. Centrifuge all samples at 4000rpm for 5 minutes at 4.0°C, Transfer the samples to autosampler vials for analysis.

Data Processing:

Acquire the data and calculate the concentration of samples using linear regression analysis with weighing factor (1/ x * x). Concentrations are back calculated using the formulae y= m x + c, where y is the area ratio, m is the slope of curve, c is intercept of curve and x is the unknown sample concentration.

RESULTS AND DISCUSSION:

Method development:

Method development was initiated, aqueous and extracted CC standards (K₂EDTA as an anticoagulant) (5 pg/ml to 10ng/ml) using LLE technique (ethyl acetate with basification) and found CC acceptable with LOQ area of ~2000 on API4000Qtrap, Acetonitril and 10 mmol ammonimum acetate was used as mobile phase in the ratio of 80:20 with 30µl injection on Inertsil ODS 150mm column, MRM was 242.05/206.1. Procedure tested and found acceptable over small duration but method performance is found lower over long period (more than 50 injections). LLE replaced with SPE and found suitable with no drop in the response but LLOQ response is between 1000 to 2000 hence we thought to introduce another daughter ion to use sum of multiple ions to get area for Std 1 minimum 2000. Blank chromatograms were presented as Figure 1 and 2.

Fig.1: Moxonidine Blank Chromatogram

Fig. 2: ISTD Blank Chromatogram

Selectivity of the method is tested using 6 plasma lots processed as blank and LLOQ and compared the blank response with that of LLOQ, no endogenous or exogenous interferences are found at the retention times. LLOQ chromatogram is presented as figure 3.

Fig. 3: Moxonidine LLOQ Chromatogram

Matrix factor for the method has been established using LQC and HQC level samples prepared in post extracted blanks and compared with that of unextracted pure samples, matrix factor found between 0.85 to 1.15 with maximum internal standard normalized matrix factor %CV of 11.5.

Intra and Inter day precision and accuracy along with ruggedness was tested by analyzing three precision and accuracy batches. Intra and Inter Precision and accuracy found acceptable. Recovery of the method found reproducible and consistent over the entire calibration curve range. ULOQ and ISTD chromatograms were presented as figure 4 and 5.

Fig. 4: Moxonidine ULOQ Chromatogram

Fig. 5: ISTD Chromatogram

Table 2: Precision and Accuracy results

Description	LLOQQC	LQC	MQC	HQC
Nominal	11.019	29.668	4262.66	7750.291
Observed	11.179	28.603	4370.51	7948.344
% Accuracy	101.5	96.4	102.5	102.6
% Precision	11	5	8	2

Calculations are done for simple weighing factor and regression selection using three calibration curves data and linear regression with 1/X²found to be simple, so was selected.

Stability of the method is tested different conditions.

Table 3: Stability durations

Stability Condition	Duration
Bench top stability at ambient temperature	6 hours
Freeze-Thaw stability	4 cycles
Wet Extract stability	25 hours
Long term stability in plasma	24 days
Autosampler stability	28 hours

Application to bioequivalence study sample analysis:

The above described method has been successfully applied to analyze a bioequivalence study consist of 12 volunteers dosed with 25mg of moxonidine as per IEC approved protocol. Screening was conducted to select volunteers as per inclusion and exclusion criteria, volunteers dosed after 10hours fasting in a crossover study design. Blood samples are collected at regular intervals. A total of 360 samples were collected before and after administration of Moxonidine, plasma separated and stored at -70⁰C. These samples were analyzed under calibration curve along with QC samples. CC and QC during study analysis showed accuracy and precision within the acceptance limits stated in EMA guideline on Bioanalytical method validation. Study analysis is conducted under GLP regulated environment. Non compartmental pharmacokinetic analysis is conducted, Mean Graph is presented as figure 6.

Fig. 6: Pharmacokinetic mean graph for Moxonidine Tablets 25 mg test (PI) vs Reference formulation (PII).

CONCLUSION:

Moxonidine is a low molecular weight basic polar drug. Basic functional groups are active for extraction and ionization. Because of the drug polar in nature, we got matrix effect problems but clonidine (ISTD) is compensating those, IS normalized matrix factor is within limits. Method validation found acceptable. Method can be used for intended purpose.

Successfully developed and validated a simple, rapid Bioanalytical method for the estimation of Moxonidine in plasma by LC-MS/MS. Sample extraction using solid phase extraction while separation was achieved on hypurity C8 column, acquired in MRM mode and quantified using analyst 1.6.2 software. Method found selective, no endogenous or exogenous interfences found either at analyte or internal standard retention times. Matrix factor ratio found around 1 whereas recoverys found more than 40 % with within 20% CV. Signal to noise ratio found always more than 5. Precision and accuracy, ruggedness and all stability experiments found acceptable. Method is successfully applied to analyze unknown samples upto 360 and found reproducibility as well.

ACKNOWLEDGEMENTS:

The authors would like to thank the management and our mentor Dr Raman Batheja of VerGo clinicals, Goa, India, for supporting to conduct the research work.

CONFLICT OF INTEREST:

The authors confirm that they do not have any conflict of interest in this report.

REFERENCES:

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5. Stephen D Wise, Clark Chan, Hans G Schaefer, Minxia M He, Isabelle J Pouliquen, Malcolm I Mitchell: Br J Clin Pharmacol, 2002, 54(3), 251–254.

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Received on 24.02.2020 Modified on 30.04.2020

Research J. Pharm. and Tech. 2020; 13(12):5773-5776.

DOI: 10.5958/0974-360X.2020.01006.9

Name	Q1 Mass (amu)	Q3 Mass (amu)	Dwell (msec)	DP (V)	EP (V)	CE (V)	CXP (V)
Moxonidine	242.0	206.1	200	88	10	30	13
Moxonidine	242.0	199.0	200	88	10	30	13
Clonidine	230.1	213.2	200	101	10	40	4
Column	Mobile Phase			Flow Rate		Injection Volume
Hypurity C8, 100 x 4.6 mm	Acetonitrile: 10mmol Ammonium Acetate) (85:15)			1.00mL/min		30µl