Method development and validation for estimation of Felodipine in human plasma by LCMS/MS

 

Srinivas Reddy, Jibin V. Joseph, Aravind Kumar, Arindam Mukhopadhyay*, Saral Thangam

Norwich Clinical Services Pvt. Ltd., 147/F, 8th Main, 3rd Block, Koramangala, Bangalore– 560034

*Corresponding Author E-mail: arindam.mukhopadhyay@norwichclinical.com

 

After extraction from plasma by liquid – liquid extraction method, Felodipine and FelodipineD5, (IS), were separated on a Reverse phase chromatography using the mobile phase mixture of ammonium acetate and methanol at a flow rate of 1.0 ml/min. The analytes were detected in API 4000 Mass spectrometer in the positive atmospheric pressure chemical Ionization (APCI) mode with multiple reactions monitoring (MRM). The MRM transitions were monitored by following m/z for parent ion 384.0 & daughter ion 338.0 (Felodipine), and m/z 389.1 & daughter 338.1 (Felodipine D5, IS). A linear calibration plot of Felodipine was achieved in the concentration ranges of 0.105 ng/ml to 20.000 ng/ml.  Mean recovery was 69.4%.This method was fully validated for specificity, precision, accuracy, reproducibility and other criteria as per regulations.

 

 

INTRODUCTION:

Felodipine, RS ethyl, methyl, 4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5- dicarboxylate with Molecular formula C₁₈H₁₉C_l2NO₄, is an antihypertensive drug belonging to second generation calcium channel blocker (CCB). Felodipine have anti-anginal activity preventing calcium from being released within muscle cells of the small arteries and thereby causing the muscle to relax and the arteries to dilate or expand (1 - 5).

 

Felodipine is slightly yellowish, crystalline powder with melting point 145⁰C. It is photosensitive and insoluble in water but is freely soluble in dichloromethane and ethanol. Felodipine is orally absorbed and undergoes extensive first pass metabolism. Several methods including HPLC and   LC MS methods have been reported for quantification of Felodipine in plasma (6 – 10). However, most of them are either lacking full validation procedures outlined by regulatory agencies or use SPE procedure. The present investigation reports a simple, rapid, sensitive, and reproducible atmospheric pressure chemical Ionization (APCI) LC MS method for analysis of Felodipine in plasma, using Felodipine D5 as internal standard (IS).

 

This method also used a cost effective and simple LLE method for sample extraction instead of costlier and cumbersome SPE. This method has been validated as per the FDA regulations and can also be used for estimation of this drug for pharmacokinetic analysis and other studies.

 

Experimental:

Materials and Reagents

Felodipine (USP) was purchased from LGC Promochem whereas deuterated Felodipine, used as an internal standard was fromVivan Life Sciences. HPLC grade methanol and acetonitrile were obtained from E Merck (India). All other chemicals of highest purity grade were locally purchased.  Milli Q water (Millipore (USA)) was used throughout the procedure.

K₂ EDTA containing human blood was collected in-house from healthy volunteers. Plasma was separated by centrifuging at 3000 rpm for 10 min at 4°C.

 

Preparation of Calibration standard samples:

Stock solutions (1mg/ml) of Felodipine were prepared in methanol. Concentration was then corrected using the potency and actual amount weighed. Working solutions (5.242 ng/ml to 1000.000 ng/ml) were prepared by serial dilution of the stock solution by methanol: water (1:1, v/v). Sodium vapour lamp was used during the whole procedure as a special precaution due to the light sensitivity of Felodipine.

Similarly for internal standard (IS), stock solution (1mg/ml) of Deuterated Felodipine (Felodipine D5) was prepared in methanol and corrected final concentration was obtained using the potency and amount weighed. Working solution (100ng/ml) was then prepared from this stock solution by serial dilution using methanol: water (1:1, v/v) as diluent. All solutions were stored in refrigeratorat 2-8ºC with due protection from light until analysis.

 

Calibration standards of concentration range from0.105 ng/ml to 20.000ng/ml were prepared by adding 980ml of blank plasma to 20ml of respective working solution and stored at – 70ºC.

 

Preparation of Quality Control Samples:

Working solutions (5.315 ng/ml to 760.000 ng/ml) were prepared by serial dilution of the stock solution by methanol: water (1:1, v/v). Quality control samples, marked as LOQQC, LQC, MQC and HQC containing 0.106ng /ml, 0.287ng/ml, 6.840 ng/ml and 15.200 ng/ml of Felodipine respectively, were prepared by adding 980ml of blank plasma to 20ml of respective working solution and stored at -70°C.

 

Sample extraction:

After thawing the sample at room temperature it was vortexed to ensure complete mixing of the contents. 50 ml of IS was added to each of these analyte spiked plasma (400ml) except blank and vortexed. 400ml of sample was transferred to a new RIA vial and added 100µl of 100 mM disodium hydrogen phosphate dihydrate and vortexed. After addition of 2.5ml of TBME all samples were kept in vibramax for 10 min at 2500 RPM. The samples were then centrifuged at 4000 RPM for 5 min at 4°C. 2ml of supernatant was transferred into the labeled RIA vial, dried at 40^oC in nitrogen evaporator, followed by reconstitution of the dried samples with 500µl of mobile phase. After vortexing the samples were centrifuged at 11000 RPM for 5 mins at 4°C. Supernatant was transferred to labeled HPLC vials and loaded into auto sampler. 20ml was injected to LCMS/MS for analysis.

 

Chromatography:

The drug was separated on a Zorbax SB, C8, 4.6 X 50 mm column with particle size 3.5m (Agilent) using the mobile phase [10mM ammonium acetate (pH 4.0±0.2): Methanol::30:70 v/v] at a flow rate of 1.0 ml/min in Waters UPLC attached to API 4000 Mass spectrometer (Applied Biosystems, USA) using  positive Atmospheric Pressure chemical Ionization mode. The column oven temperature was maintained at 50⁰C and the run time was 3.00 min. The analytes were detected on mass spectrometer operating in the multiple reaction monitoring (MRM) modes. The following precursor à product ion transition was monitored for MRM transitions: m/z 384.0 à 338.0 (Felodipine) and m/z 389.1  à 338.1 (Felodipine D5) with a dwell time of 200 msec. The APCI source temperature was maintained at 300⁰C. Data were acquired and processed with Analyst software 1.5.1.

 

Matrix Factor:

Six replicates of aqueous standard containing analyte at LQC concentration and the intended internal standard concentration were injected. Individual analyte area response and IS area response of each post extracted sample were compared with the mean analyte area response and mean IS area response of the aqueous standard respectively. Similar experiment was performed with HQC concentration of analyte.

 

Matrix factor is calculated as the ratio of area response in presence and absence of matrix.

 

Result and Discussion:

Specificity and Selectivity:

For selectivity analysis, eight different lots of plasma including one hemolysed and one lipemic lots were spiked with analytes and internal standard. Interference at the retention times of analytes and IS was evaluated by comparing peak area response with that of blank plasma. Signal to noise ratio for all lots was more than 5.0 indicating the method is selective for Felodipine. Retention times for Felodipine and Felodipine D5 were 1.86 min and 1.84 min, respectively (Fig.1a and  b). No interfering peaks were observed in the blank at the retention times corresponding to drug and IS indicating that the procedure is specific to Felodipine.

 

Similarly, no matrix effect was found while analyzing the human plasma samples, (Table 1).

 

 

Table 1: Matrix Effect for estimation of Felodipine

Matrix ID	Analyte Area In Absence of Matrix	Analyte Area in Presence of Matrix	Matrix Factor For Analyte	IS Area in Absence of Matrix	IS Area in Presence of Matrix	Matrix Factor for IS
PL_161	7042	7616	104.53	299845	305436	103.09
PL_162	6994	7432	102.01	297986	303575	102.47
PL_163	7141	7520	103.21	289817	311179	105.03
PL_164	7580	7532	103.38	299630	311368	105.10
PL_165	7524	7791	106.93	297279	313579	105.84
PL_166	7434	7727	106.06	300268	315922	106.63
HPL_135		7920	108.70		314222	106.06
LPL_137		7568	103.87		316724	106.90

 

 

 

Fig 1a. Felodipine

 

Fig 1 b: Felodipine D5 (IS)

Linearity of the Calibration Plot:

Calibration plots of Felodipine showed that the calibrations are linear in the concentration ranges of 0.105 ng/ml to 20.000 ng/ml with a correlation coefficient (r) of 0.99. Limit of detection was found to be 0.026ng/ml with a signal to noise ratio of more than 3.0.

 

Precision & Accuracy:

Intra- or inter- day precision and accuracy were determined by six replicate analysis of LOQQC, LQC, MQC and HQC samples. Intraday precisions were ranged from 0.26% to 12.34% for Felodipine which were within acceptable limit (Table 2). Similarly, for inter-day batch precision %CV ranged from 1.65% to 10.20% for Felodipine was also within accepted limit (£ 20% at LOQQC and £15% for others).

Mean accuracy for intraday batch ranged from 91.03% to 112.66% for Felodipine which are within acceptable limit (Table 2). Similarly, for inter-day batch accuracy ranged from 99.03% to 103.75% which were also within accepted limit (£ 20% at LOQQC and £15% for others).

 

Recovery:

Absolute recovery percentage was determined by comparing the mean peak area of Felodipine obtained by injecting 6 extracted samples of LQC, MQC and HQC with the mean peak area obtained by injection of respective aqueous standard solutions. Mean percentage recovery was 69.04 with mean % CV of 3.63 (Table 3).

 

 

Table 2: Accuracy and precision of analysis of Felodipine in the quality-control samples

 

Table 3: Recovery of Felodipine from biological matrix

LQC			MQC			HQC
Unextracted area (n=6)     0.306ng/mL	Extracted area (n=6)     4.412ng/mL	Mean percentage recovery	Unextracted area (n=6)     7.654ng/mL	Extracted area (n=6)     7.654ng/mL	Mean percentage recovery	Unextracted area (n=6)     15.308ng/mL	Extracted  area (n=6)   15.308 ng/mL	Mean percentage recovery
8626	6190	71.76	224532	153842	68.52	470290	314344	66.84

 

Table 4: Stability of felodipine in biological matrix

Stability Check	Samples	Nominal Conc.	Observed Conc.	%cv	% Stability
Bench Top for (11hrs)	LQC (n=6)	0.306	0.291	4.89	95.10
	HQC(n=6)	15.308	14.576	0.97	95.22
Freeze Thaw (4 cycles)	LQC (n=6)	0.306	0.294	6.92	96.08
	HQC(n=6)	15.308	14.309	1.21	93.47
In-Auto-sampler(31hr)	LQC (n=6)	0.306	0.278	0.92	90.85
	HQC(n=6)	15.308	13.884	1.01	90.70
Wet Extract (3hr)	LQC (n=6)	0.306	0.325	4.06	106.21
	HQC(n=6)	15.308	15.079	2.83	98.50

 

Table 5: Stability of Aqueous FelodipineSolutions

Stability Check	Samples	Fresh Stock (Avg. Area)	Stability Stock (Avg. Area)	%cv	% Stability
Short Term Stock Solution Stability (25hr)	Analyte(n=6)	219486(1267.9590µg/ml)	206733(1275.6436µg/ml)	0.81	94.76
	Internal Standard (n=6)	314721(1022.8194µg/ml)	298622(1016.3396µg/ml)	1.02	95.49
Short Term Working solution Stability (25hr)	Analyte (n=6)	219486(380.388ng/ml)	211836(382.692ng/ml)	2.31	91.70
	Internal Standard (n=6)	314721(102.282ng/ml)	304096(101.634ng/ml)	2.50	97.24
Long Term Stock Stability (15 Days)	Analyte(n=6)	189256(1275.6436µg/ml)	203919(1297.8990µg/ml)	1.15	105.90
	Internal Standard(n=6)	302811(1016.3396µg/ml)	319741(1016.3396µg/ml)	1.46	105.59

 

 

Stability:

Short – Term/bench - top stability

To check whether the sample is stable during analysis, six aliquots of LQC & HQC samples were thawed and kept at room temperature under yellow monochromatic light for 11 hours, which has been decided based on the time required for analysis. The samples were then processed and analyzed as mentioned above. No significant differences were noticed when these results were compared with those obtained from the freshly spiked samples indicating that Felodipine was stable at room temperature (Table 4).

 

Auto sampler stability

The stability of the processed samples in the auto sampler during analysis was determined by using six aliquots of LQC, HQC samples. The stability of Felodipine was assessed for 31 hours, the expected run time for batches of validation samples. The result was then compared with that of freshly spiked samples. For IS in-injector stability, the IS/analyte area ratio of MQC stored in auto sampler for 31 hours was compared against freshly prepared MQC samples .No significant difference in the results indicated that the analytes and IS are stable for at least 31 hour in the auto sampler (Table 4).

 

Freeze – Thaw stability

Analyte stability was determined after four freeze thaw cycles for six aliquots of each of the LQC and HQC. The samples were stored below – 70⁰C for 24h and then allowed to thaw at room temperature unassisted. After complete thawing, the samples were again stored at same temperature (– 70⁰C) for 12h. The freeze thaw cycle was repeated another three times before analyzing the samples. No differences were noticed when the results were compared with the fresh QC samples indicating the stability of Felodipine in K₂EDTA human plasma for four freeze thaw cycles (Table 4).

 

Wet Extract stability

To check whether the sample is stable after processing, six aliquots of LQC & HQC samples were processed and kept at room temperature under yellow monochromatic light for 3 hours. The samples were then analyzed as mentioned above. No significant differences were noticed when these results were compared with those obtained from the fresh QC samples indicating that processed samples of Felodipine was stable at room temperature (Table 4).

 

Short term stock solution stability

To ensure that analyteis stable in appropriate solution for a short period of time at room temperature, the stability of stock and working solutions of Felodipine was evaluated at room temperature for 25 hours under yellow monochromatic light. There were no significant changes in stabilities of the stock and working solutions on keeping at room temperature for 25 hours (Table 5).

 

Long term stock solution stability

To evaluate the stability of analyte in appropriate solution for a long period of time under storage condition (2 – 8⁰C), the stability of stock solution of Felodipine was evaluated at 2 – 8⁰C for 15 days. Table 5 indicates that the stock solution was stable during the storage.

 

CONCLUSION:

A simple, accurate, precise, sensitive and reproducible LCMS/MS method has been developed and validated for the determination of Felodipine. The atmospheric pressure chemical Ionization technique is used to overcome the matrix effect. The sample extraction procedure described here is LLE method which is not only a simpler method compared to other available extraction methods but also produces cleaner samples with no matrix effect. The method is applied successfully to quantify Felodipine in the pharmacokinetic study.

 

ACKNOWLEDGEMENT:

The authors sincerely thank the management of Norwich Clinical Services for providing the opportunity to complete the project.

 

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Received on 13.02.2013         Modified on 24.02.2013

Accepted on 06.03.2013         © AJRC All right reserved