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Development and validation of a liquid chromatography mass spectrometry method for the determination of donepezil in human plasma  

2018-03-28 04:30:57|  分类: 二分五性三码一谱 |  标签: |举报 |字号 订阅

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PrakashKatakamacRama RaoKalakuntlabShanta KumariAdikiaBabu RaoChanduc

https://doi.org/10.1016/j.jopr.2013.08.021  Journal of Pharmacy Research  Volume 7, Issue 8, August 2013, Pages 720-726
Abstract  Aim A selective, and sensitive LC–MS/MS method has been developed and validated for quantification of donepezil in human plasma using donepezil D7 as an internal standard (IS).

Methods

The analyte and IS were extracted by liquid–liquid extraction using dichloromethane and hexane mixture and separated by isocratic elution on C18 analytical column with 0.1% formic acid and methanol in the ratio of 70:30 (flow rate of 1 ml/min) as the mobile phase in the positive ion mode. Multiple Reaction Monitoring transitions for donepezil and internal standard are 380.2/91.2 and 387.2/98.2 respectively.

Results

The lower limit of quantification was 50 pg/ml with the linearity range of 50 pg/ml–25,000 pg/ml and the method was validated as per international regulatory guidelines for its selectivity, stability, accuracy, precision, and recovery.

Conclusion

The method can be readily applicable to pharmacokinetic and bioequivalence studies to support different regulatory submissions.

1. Introduction

Donepezil (Fig. 1) is a piperidine-based, reversible inhibitor of the enzyme acetylcholinesterase. Donepezil is indicated for symptomatic treatment of patients with mild, moderate and severe dementia of the Alzheimer's type. Alzheimer's disease is a neurodegenerative disorder characterized by progressive loss of memory followed by complete dementia. It accounts for 50% of dementia cases.1 A consistent pathological change in Alzheimer's disease is the degeneration of cholinergic neuronal pathways that project from the basal forebrain to the cerebral cortex and hippocampus. The resulting hypofunction of the cholinergic systems is thought to account for some of the clinical manifestations of dementia. Donepezil is postulated to exert its therapeutic effect by enhancing cholinergic function and acetylcholine levels of the brain. This is accomplished by increasing the concentration of acetylcholine through reversible inhibition of its hydrolysis by acetylcholinesterase.


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Fig. 1. Donepezil.



The recommended initial dose of donepezil is 5 mg taken once daily. Donepezil is well absorbed with a relative oral bioavailability of 100% and reaches peak plasma concentrations (Cmax) approximately 3–4 h after dose administration. In humans, donepezil is metabolized mainly by the hepatic cytochrome P-450 2D6 and 3A4 isozymes.2 Elimination of donepezil from the blood is characterized by a dose independent elimination half-life of about 70 h.3,4 Because plasma donepezil concentrations are related linearly to acetylcholinesterase inhibition,5 plasma donepezil concentration is a useful tool to predict donepezil efficacy.

In the literature, methods have been reported for the quantification of donepezil in biological fluids. Methods are reported for the quantification of donepezil from biological matrix using high-performance liquid chromatography (HPLC) equipped with an ultraviolet detector,2,3fluorescence detector4 and mass spectrometric1,6,7 detector. Methods are also reported for the quantification of enantiomers of donepezil from human plasma.8–10 Other methods are reported with estimation of donepezil in plasma by capillary electrophoresis,11 hydrophilic interaction chromatography-tandem mass spectrometry,12 direct measurement,13 automated extraction.14The HPLC methods used to determine donepezil in human plasma are insensitive because of the lower limit of quantification (LOQ of >1.0 ng/ml). Some of the reported methods1,4,6,10,13,14utilized analogue internal standards like diphenhydramine, lidocaine, pindolol, loratadine, escitalopram, etc. and are validated with different calibration curve ranges for the estimation of donepezil from rat plasma, human plasma and other biological fluids. Usage of labelled internal standards is recommended during the estimation of compounds from the biological matrices to minimize the matrix effects associated with the mass spectrometric detection.

Bioequivalence and/or pharmacokinetic studies become an integral part of generic drug applications and a simple, sensitive, reproducible validated bioanalytical method should be used for the quantification of intended analyte. Bioequivalence studies for the donepezil needs to be performed with the dosage of 10 mg and 23 mg tablets to support the generic abbreviated new drug applications. For the pharmacokinetic and bioequivalence studies, quantification of donepezil was sufficient and quantification of its metabolites shall not be required. During the bioequivalence studies, appropriate lower limit of quantification needs to be used to appropriately characterize the concentration profile including the elimination phase.

The motive of the present method was to develop the method with the lower limit of quantification of 50 pg/ml and with minimal sample volume using simple and cost effective liquid–liquid extraction technique. The developed method was validated as per the current international regulatory guidelines on bioanalytical method validation. The method can be readily applicable for usage during the bioequivalence evaluation of various generic formulations for submission as part of abbreviated new drug applications.


2. Materials and methods

2.1. Chemicals

Donepezil reference standard was procured as a gift sample from a Pharma company and HPLC grade methanol, acetonitrile were commercially procured and all other chemicals were of analytical grade.


2.2. Preparation of solutions

0.01 N hydrochloric acid was prepared by diluting 0.1 ml of hydrochloric acid to 1000 ml in a volumetric flask with milli Q water. Mixture of dichloromethane and hexane was prepared by mixing one part of dichloromethane and four parts of hexane. 1% formic acid was prepared by adding 10 ml of formic acid to a 1000 ml volumetric flask and made up the volume with milli Q water and similarly 0.1% formic acid solution was prepared by adding 1 ml of formic acid to a 1000 ml volumetric flask and made up the volume with milli Q water. 50% methanol was prepared by mixing 500 ml of methanol and 500 ml of water in a reagent bottle. Rinsing solution which is used for auto sampler wash was prepared by mixing 0.1% formic acid and methanol in the ratio of 80:20. Mobile phase consisting of 0.1% formic acid and methanol mixture (70:30) was prepared by mixing 700 ml of 0.1% formic acid with 300 ml of methanol.


2.3. Preparation of standards

Donepezil and donepezil D7 stock solutions were prepared at a concentration of 0.1 mg/ml by dissolving in 0.01 N hydrochloric acid solution and the stock solutions were stored in the refrigerator. Spiking solutions of donepezil for the preparation of calibration standards and quality control samples were prepared in mobile phase and spiked in to the plasma at the ratio of 1:50. The calibration curve from 50 to 25,000 pg/ml was generated using ten calibration standards at the concentrations of 50 pg/ml (STD 1), 100 pg/ml (STD 2), 200 pg/ml (STD 3), 500 pg/ml (STD 4), 2500 pg/ml (STD 5), 5000 pg/ml (STD 6), 10,000 pg/ml (STD 7), 15,000 pg/ml (STD 8), 20,000 pg/ml (STD 9), 25,000 pg/ml (STD 10). The quality control samples were prepared at the concentrations of 50 pg/ml (LLOQQC), 150 pg/ml (LQC), 9000 pg/ml (MQC) and 18,000 pg/ml (HQC). The bulk spiked calibration standards and quality control samples were stored in the freezer. Internal standard dilution was prepared at a concentration of 3000 pg/ml using mobile phase.


2.4. Sample preparation and extraction

Donepezil from the plasma was extracted using liquid–liquid extraction technique. Plasma aliquot of 0.3 ml (300 μl) was added to the polypropylene tube containing 50 μl of internal standard dilution and vortexed the tubes. 0.5 ml of 1 N sodium hydroxide solution was added and vortexed for thorough mixing. To vortexed sample added 5 ml of dichloromethane and hexane mixture and tumble the tubes for about 10–15 min. After tumbling the tubes are centrifuged at 3000 rpm for about 5–10 min. After centrifugation, the supernatant was transferred into the polypropylene tubes and evaporated to dryness under the stream of nitrogen at 40 °C. After evaporation, the tubes are reconstituted with 0.15 ml of mobile phase and transferred to auto sampler vials for injection.


2.5. Chromatographic conditions

HPLC coupled with Mass Spectrometer (LC–MS/MS) with the C18 column (4.6 × 75 mm, 3.5 μl) was used and the m/z of 380.2/91.2 and 387.3/98.2 were used in Multiple Reaction Monitoring (MRM) mode with turbo ion spray in positive mode for the quantification of donepezil and internal standard respectively. The other mass spectrometric conditions are optimized for reproducible response. The mobile phase used was 0.1% formic acid and methanol in the ratio of 70:30.


2.6. Validation

The method performance was evaluated for selectivity, accuracy, precision, linearity, and robustness, stability during various stress conditions including bench top stability, freeze thaw stability, auto sampler stability, stability of stock solutions etc., dilution integrity and recovery.


2.7. Selectivity

Selectivity was evaluated by extracting different blank plasma samples. The absence of interfering peaks at the retention time of analyte or internal standard was considered as evidence for selectivity.


2.8. Linearity

Calibration curves were constructed after evaluating the linear regression for the best fit using weighing of none, 1/x and 1/x2 for the calibration curve range of 50.1–25,052.5 pg/ml.


2.9. Precision and accuracy

For precision and accuracy studies, samples were prepared at four concentration levels, limit of quantification (LOQQC), low (LQC), medium (MQC) and high (HQC) quality controls. Corresponding to 50.1, 150.3, 9017.1 and 18,034.2 pg/ml respectively with six replicates each. Precision and accuracy was evaluated at inter and intraday.


2.10. Recovery

Recovery of analyte was evaluated by comparing the donepezil and internal standard response in extracted samples versus equivalent aqueous samples. Recovery was evaluated at three levels of quality control samples (LQC, MQC and HQC levels). The mean recovery of analyte and internal standard was evaluated.


2.11. Matrix effect

Matrix effect of was evaluated by comparing the donepezil and internal standard response in aqueous samples versus post extracted samples. Matrix factor of analyte and internal standard were calculated and subsequently internal standard normalized matrix factor was also calculated.


2.12. Dilution integrity

Dilution integrity was evaluated by diluting the sample having the concentration of approx. 35,000 pg/ml (approx. two times of HQC) with 1/5 and 1/10 dilutions and quantified against the calibration curve to evaluate the ability to dilute the pharmacokinetic samples.


2.13. Stability studies

The stability of the donepezil in solutions and plasma samples was also evaluated during method validation. Donepezil stability was evaluated using two concentration levels (low and high quality control, corresponding to 50.1 and 18,034.2 pg/ml respectively). The stability of donepezil was also evaluated in post-extracted samples kept in the auto sampler at 10 °C as well as in plasma samples kept at freezer and after being stressed to freeze–thawing cycles (24 h each cycle). All samples described above were quantified using fresh calibration curve and compared to freshly prepared quality control samples at the same concentration level.


3. Results and discussion

3.1. Chromatographic optimization

Liquid chromatography coupled with the mass spectrometer (LC–MS/MS) has now become a universally acceptable technique for the estimation of drugs from the biological fluids as part of bioequivalence evaluations. Donepezil and internal standard were scanned in the positive mode for the parent ion and reproducible daughter ion and the m/z ratio of 380.2/91.2 and 387.3/98.2 respectively were selected for donepezil and internal standard. The quantification was performed in Multiple Reaction Monitoring (MRM) mode in analyst software. The compound specific mass spectrometric parameters are optimized to produce the reproducible responses for the analyte and internal standard. Chromatographic conditions are optimized to achieve good resolution and symmetric peak shape for the analyte at the lower level of quantification. The chromatographic conditions like flow rate (1.0 ml/min) and column (C18 column) conditions were also optimized with the runtime of 4 min. The analyte and internal standard were quantified at 1.8 min. Other conditions are optimized for the reproducible quantification method.


3.2. Sample preparation

Liquid–liquid extraction technique was chosen for the simple and cost effective extraction procedure and the conditions are optimized to yield cleaner extract of the sample to avoid the quantification issues with the LCMSMS. Protein precipitation with acetonitrile was tried but the recovery was found to be low. Organic solvent mixture consisting of dichloromethane and hexane was yielded good recovery and better chromatography compared to individual solvents. Sample volume of 300 μl was optimized to have the sensitivity and quantifiable and acceptable peak shape at the lower limit of quantification of 50 pg/ml. Lesser sample volumes are also attempted but the peak shape and response at the lower limit of quantification are not acceptable with respect to signal to noise ratio. The quality control samples were prepared at the concentrations specified in the bioanalytical method validation guidelines. The LOQQC was prepared at approximately same concentration of lowest calibration standard. The LQC was prepared at the concentration less than three times of lowest calibration standard. MQC concentration was prepared at approximately 35% of the highest calibration standard. HQC concentration was prepared at the concentration of approximately 70% of the highest calibration standard.


3.3. Selectivity

The LCMSMS method was selective for the intended analyte since the quantification is based on the mass to charge ratio of parent as well as product ion in MRM transition mode which are selective and specific. The selectivity was also established for the blank plasma lots with the acceptance criteria of analyte response and internal standard response observed in the blank plasma samples shall be less than 20% and 5% of the analyte and internal standard response of LLOQ sample. No interference was observed for the blank plasma lots at the analyte and internal standard retention times. Figs. 2–4 represent the chromatogram of blank plasma sample and lowest calibration standard and highest calibration standards respectively.


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Fig. 2. Representative chromatogram of blank plasma sample.



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Fig. 3. Representative chromatogram of lowest calibration standard.



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Fig. 4. Representative chromatogram of highest calibration standard.




3.4. Linearity

Linearity was demonstrated from 50.1 to 25,052.5 pg/ml. Table 1 shows data from calibration curves analysed for the evaluation of precision and accuracy during different days. The calibration curve includes ten calibration standards which are distributed throughout the calibration range. Correlation coefficient was considered for the evaluation of goodness fit. The average correlation coefficient was found to be 0.9987 with goodness of fit.


Table 1. Between and within batch precision and accuracy of QC samples.


LOQQC LQC MQC HQC
Nominal concentration 50.1 150.3 9017.1 18,034.2
Precision and accuracy batch-1 51.8 153.2 8794.6 17,132.5
51.6 151.6 9092.1 17,870.4
49.7 147.5 8873.7 17,612.5
45.7 135.1 8739.4 17,304.0
48.7 133.3 8934.2 17,199.6
52.6 144.3 8960.6 17,915.9
Precision and accuracy batch-2 52.8 147.0 9176.4 21,630.7
55.0 154.0 9208.6 17,323.4
57.1 145.5 8810.3 18,348.8
51.8 161.1 8429.8 17,067.9
47.9 151.9 8524.0 17,208.8
51.0 144.9 8681.1 16,893.2
Precision and accuracy batch-3 55.1 153.4 9977.4 17,615.2
50.2 150.7 9624.2 18,763.5
54.5 149.4 9290.3 18,294.9
49.9 151.6 8961.8 18,724.5
50.3 158.3 8864.2 18,539.9
51.5 164.0 8814.9 19,140.2
Global precision and accuracy
Average 51.51 149.82 8986.53 18,032.55
Standard deviation 2.789 7.783 374.523 1123.237
% CV 5.4 5.2 4.2 6.2
% nominal 102.8 99.7 99.7 100.0


3.5. Precision and accuracy

Precision and accuracy was evaluated by analysing three precision and accuracy batches. Each precision and accuracy batch consists of calibration curve and six replicates of LOQQC, LQC, MQC and HQC. Precision and accuracy was evaluated both inter and intra batches. The intraday and interday precision and accuracy of the method for each donepezil concentration level (50.1, 150.3, 9017.1 and 18,034.2 pg/ml) are presented in Table 1. The mean accuracy for each concentration level ranged from 99.7 to 102.8 and the mean precision for each concentration level ranged from 4.2 to 6.2.


3.6. Recovery

The recovery was evaluated by comparing response of extracted and unextracted samples. Extracted samples include six replicates of extracted LQC, MQC and HQC samples. Unextracted samples included the aqueous solutions equivalent to extracted samples. Internal standard recovery was evaluated in the same manner at MQC level. The average recovery for donepezil in plasma was ranged from 42.8 to 56.0% for the low, medium and high quality control samples respectively with an average of 51.6%. The average recovery of the internal standard was 47.1%.


3.7. Matrix effect

Matrix effect of was evaluated in six different blank plasma lots. Post spiked samples are prepared by adding the spiking solution to the blank plasma samples processed till the evaporation step. The post spiked samples are compared against the equivalent aqueous concentrations. The mean internal standard normalized matrix factor was found to be close to 1 indicating that matrix effect does not influence the method performance.


3.8. Stability studies

Stability studies were performed to evaluate the stability of donepezil both in aqueous solution and in plasma after exposing to various stress conditions. The stability studies performed include stock solution stability of donepezil and donepezil D7 in stock solution, stock dilution stability of donepezil in dilutions, bench top stability in plasma, freeze thaw stability in plasma, long term storage stability in plasma, and auto sampler stability of processed samples. All stability evaluations were performed as per international regulatory guidelines.


Donepezil and donepezil D7 stock solutions (0.1 mg/ml) remained stable when stored at refrigerator conditions for 7 days including the storage at room temperature for 8 h. Donepezil was stable in plasma samples when stored at room temperature for 19 h. Donepezil was found to be stable for three freeze and thaw cycles. Donepezil was stable and did not show any degradation when stored in the freezer for 96 days. Donepezil in the processed samples was stable for 82 h when stored in the auto sampler at 10 °C. The method characteristics are represented in Table 2.


Table 2. Method characteristics.


Analyte Donepezil
Internal standard Donepezil D7
Method description Liquid–liquid extraction with LCMSMS detection
Regression model Linear regression with 1/conc2 weighing
Analysis method Peak area ratios
Limit of quantification 50.0 pg/ml
Selectivity No interference from the endogenous matrix components
Recovery of analyte 51.6%
Recovery of internal standard 47.1%
Linearity range 50.1–25,052.5 pg/ml
Quality controls concentrations LLOQQC: 50.1 pg/ml
LQC: 150.3 pg/ml
MQC: 9017.1 pg/ml
HQC: 18,034.2 pg/ml
QC interday accuracy range 99.7–102.8%
QC interday precision range 4.2–6.2%
Bench top stability 19 h at room temperature
Freeze–thaw stability 3 cycles
Auto injector stability 82 h
Long term stability 96 days
Stock solution stability 7 days
Stock dilution stability 26 h
Dilution integrity 1/5 and 1/10
Re-injection reproducibility One time


4. Conclusion

We described here the development of a new, selective, precise and accurate method for the quantification of donepezil in human plasma using Liquid Chromatography Mass Spectrometric method with the simple liquid–liquid extraction technique using the less volume of plasma and is suitable for application to a pharmacokineticbioequivalence and drug interaction studies for the estimation of donepezil from plasma. The limit of quantification of the method was set to 50 pg/ml considering the dosage of donepezil administered and it is determined not only by detection technique but also by the effective clean-up of sample and thus improving the signal to noise ratio. The method reported here uses a simple and effective extraction technique with good and reproducible recovery.


Conflicts of interest

All authors have none to declare.

Acknowledgements

Authors are thankful to JPR Solutions for providing the support during publication.


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