Key word
nifedipine - HPLC-MS/MS - pharmacokinetics - acetaminophen
Abbreviations
LLE: liquid-liquid extraction
MRM: multiple reaction monitoring
SPE: solid-phase extraction
Introduction
Nifedipine, 1, 4-Dihydro-2, 6-dimethyl-4-(2-nitrophenyl)-3, 5-pyridinedicarboxylic
acid dimethyl ester ([Fig. 1a]), is a dihydropyridine calcium channel blocker and can be used for treatment of
hypertension, angina pectoris and other vascular disorders. Through physically plugging
the channel and resulting in decreases of intracellular calcium levels, inhibiting
of the contraction processes of smooth muscle cells, dilate the coronary and systemic
arteries, increase oxygen delivery to the myocardial tissue, decrease total peripheral
resistance and systemic blood pressure [1].
Fig. 1 The chemical structure of nifedipine a and acetaminophen b.
Numerous analytical methods included gas chromatography (GC), liquid chromatography
(LC) and high performance liquid chromatography (HPLC) have been reported for the
determination of nifedipine in biological samples. GC methods often utilize electron-capture
detection, flame ionization detection, nitrogen-phosphorus detection, or mass spectrometric
(MS) detection [2]
[3]
[4] to provide high sensitivity for nifedipine pharmacokinetic studies, but the method
has several drawbacks, including its thermal decomposition under GC condition and
time-consuming. LC methods without coupling with MS detection [5]
[6] were also time-consuming in sample disposition. Furthermore, the linearity range
from 10–200 ng/mL did not fulfill the requirement for a much low limit of quantitation
(LLOQ) of nifedipine pharmacokinetic studies. While the LC/MS method based on electrospray
ionization (ESI) or atmospheric pressure chemical ionization (APCI) [7]
[8]
[9] provided an LLOQ of 1.0 ng/mL, each run need a relative long time (8 min) and large
volume of plasma samples (1.0 mL). UPLC-MS/MS, with short retain time and much lower
limit of quantification [10], is expensive and not available in many clinical laboratories. The high performance
liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method developed in this
study is simple, rapid, selective, has high sensitivity and high sample throughput
relative to other methods. Several publications have reported about HPLC-MS/MS method,
as deproteinized with methanol and the calibration curves over the range of 0.5–50 ng/mL
[11] as liquid-liquid extraction with the LLOQ of 1 ng/mL [12], did not meet the requirements for sensitivity of nifedipine determination in plasma
for human pharmacokinetic studies.
Sample pretreatment has also been an item of interest for the analysis of nifedipine
in biological samples. Previous methods included solid-phase extraction (SPE), protein
precipitation and Liquid-liquid extraction (LLE). On-line SPE is expensive and complex,
especially for a large number of samples [13]. After protein precipitation, the samples remained impure and were with the risk
of blocking and poor response [14]. A liquid-liquid extraction (LLE) method with ether: n-hexane (3:1, v/v)[15], diethyl ether [12] and cyano cartridges were conditioned successively with 2 ml of methanol, 2 ml of
Milli-Q water and 2 ml of 0.01% phosphoric acid [16] have been reported, but these reagents are toxic or expensive. A simpler, less toxic,
faster and more economical method allowing for a reduction of sample manipulation
and total analysis time was required.
A faster, sensitive, simple and less toxic HPLC-MS/MS approach based on the LLE with
ethyl acetate as the extraction solvent, is more cost-effective, validated and can
be used for the determination of nifedipine in plasma and for pharmacokinetic studies.
Experimental
Chemicals and reagents
Nifedipine (Lot: 100338-200502, purity: 99.8%) and acetaminophen (Lot: 10018-0107,
purity: 99.8%) were obtained from National institute for the Control of Pharmaceutical
and Biological Products, Beijing China. Methanol (Lot: K32E12) and acetonitrile (Lot:
H30754) were HPLC grade from J. T. BAKER, USA. Ethyl acetate (Lot: H39B05, HPLC grade)
was obtained from Sinopharm Chemical Reagent Co. LTD. Ammonium acetate (Lot: 10133751,
Analytical) was obtained from the company of A Jonson Mattery. Blood plasma (Lot:
20110926) was obtained from Qilu Hospital of Shandong University. Purified water (Lot:
20120310) was obtained from the Company of Wahaha.
HPLC/MS/MS instrumentation and chromatographic conditions
The HPLC-MS/MS procedure was performed using an Agilent 1200 series HPLC and an Agilent
6410 Triple Quadrupole mass spectrometer equipped with an electrospray ionization
source (Agilent Technologies, USA). All data were analyzed by software Agilent 6410
Quantitative Analysis version analyst data processing software.
The chromatographic separation was achieved on a Diamond C18 column (150 mm×4.6 mm, 5 µm, Dikma Technologies, Beijing, china) at 25°C with a thermostated
column oven. The mobile phase was acetonitrile mixed with 5 mM ammonium acetate solution
(pH=6.62) (60:40, v/v), with a thermostated flow rate of 0.8 mL/min. The injection
volume was 10 µL.
Mass spectrometric analysis was performed in the negative ion MRM mode, with spray
gas pressure (350 Pa), protective air of nitrogen gas 9.0 L/min, dwell times (200 ms),
and capillary voltage (4000v). The fragment electric voltage, collision energy and
quantification of nifedipine and IS (acetaminophen) were achieved by monitoring the
m/z of precursor/product ions ([Table 1]). Calculations were based on peak area radios of analyte to internal standard. Concentrations
are interpolated from a linear least squares regression and calibration curve was
based on 1/concentration2 weighting for both analytes.
Table 1 Optimized mass spectrometry parameters for nifedipine and IS.
|
Analyte
|
precursor/productions
|
Fragment electric voltage
|
collision energy(eV)
|
EMV
|
|
nifedipine
|
345.1/222.2
|
100
|
5
|
400
|
|
acetaminophen
|
150.1/107.1
|
100
|
20
|
400
|
The column was washed with a 95:5 water-acetonitrile (v/v) mobile phase for 50 min
and then with acetonitrile for 50 min when every batch was finished.
Preparation of calibration standards, internal standard, and quality controls
Nifedipine is a photo labile compound, and a nitroso-pyridine derivative is formed
in solution on exposure to visible light, while a nitro-pyridine derivative is generated
under ultraviolet light [17]. Therefore, the whole process of the experiment must be operated in dark place.
A 10.2 mg aliquot of nifedipine standard and a 10.1 mg aliquot of acetaminophen were
weighed in an analytical balance and transferred to 2 A-grade 10 mL volumetric flasks,
dissolved with methanol to obtain nifedipine (1.02 mg/mL) and IS (1.01 mg/mL) mother
solutions. Primary stock solutions were diluted with the mobile phase for standard
working solutions of nifedipine (102, 10.20, 1.02 µg/mL) and IS was dissolved with
mobile phase to get a 1010 ng/mL stock solution. All solutions were stored at 4°C
and dark places, and equilibrated to room temperature before use (approximately 15 min).
The calibration curve standard and quality control (QC) samples were freshly prepared
with blank plasma by spiking with different working solutions. The calibration samples
consist of eight nonzero concentrations (0.17~102 ng/mL), and QC samples were 0.17(LLOQ),
0.42(LQC) and 6.53(MQC), 81.60 ng/mL (HQC) for nifedipine.
Plasma pre-treatment
The aim of sample pre-treatment method should remove interferences from the biological
sample and also be reproducible with a high recovery and simple procedure involving
a minimum number of working steps and less cost. A liquid-liquid extraction method
was used for the extraction of nifedipine and IS from plasma. 50 µL of IS (1010 ng/mL
acetaminophen) was mixed with 500 µL plasma sample, then 3.5 mL ethyl acetate was
added, vortex-mixed for 2 min, and centrifuged at 5 000 rpm for 5 min (2 266 g). 3 mL
organic phase was transferred to a clean tube and evaporated to dryness under gentle
stream of nitrogen gas at 35°C. Residue was reconstituted with 100 µL mobile phase,
and 10 µL was injected onto the HPLC-MS/MS for analysis.
Method Validation
The assays of nifedipine in human plasma were validated in compliance with the US
Food and Drug Administration [18] and Chinese State Food and Drug Administration guidelines for the validation of
bioanalytical methods including assay selectivity, linearity, recovery, matrix effects,
accuracy, precision and stability.
Selectivity
The specificity of the method was evaluated by comparing chromatograms of 6 different
lots of blank human plasma to identify the potential interference of endogenous substances
at the HPLC peak region (nifedipine and IS).
Calibration curves
Calibration curves were prepared at 8 different nifedipine concentrations. Each calibration
standard was injected in 5 replicates. Calibration curves were typically described
by equation y=ax+b, where y represents the peak-area ratio of nifedipine to IS, and
x represents the plasma concentration of nifedipine. The linearity of the calibration
curves was assessed by linear regression with a weighting factor of the reciprocal
of the concentration squared (1/x2). The calibrators for analytes were: 0.17, 0.42, 1.04, 2.61, 6.53, 16.32, 40.80 and
102 ng/mL.
The low limit of quantification (LLOQ) was defined as a signal-to-noise ratio greater
than 10 and evaluated by analyzing 5 replicates of spiked plasma samples at the concentration
of 0.17. The acceptance criterion for each back-calculated standard concentration
was ±15% deviation from the nominal value, while that of LLOQ was ±20%.
Recovery and matrix effect
The mean overall recovery of the nifedipine was determined by comparing the peak areas
(extracted plasma standards/post extraction plasma samples). Nifedipine were determined
by samples at 3 QC levels (0.42 ng/mL as low, 6.53 ng/mL as medium and 81.60 ng/mL
as high) with 5 replicates for each QC level. The recovery of the IS (101 ng/mL) was
determined in a similar way.
Matrix effect was investigated to ensure selectivity, precision and sensitivity that
were not compromised by the matrix screened. Blank plasma samples were extracted and
spiked with the nifedipine at 3 QC levels and IS in 5 replicates. The corresponding
peak areas were compared to those of standard solutions, and the peak area ratio was
defined as the matrix effect.
Precision and accuracy
The intra-assay precision and accuracy were estimated by analyzing 5 replicates of
nifedipine at 3 different QC levels (0.42 ng/mL, 6.53 ng/mL and 81.60 ng/mL) in human
plasma. The inter-assay precision was determined by analyzing the 3-level QC samples
on 3 consecutive days. The criteria for acceptability of data were accuracy within
85%~115% from the nominal values and a precision of within ± 15% relative standard
deviation (RSD) or CV%, but that of LQC is not supposed to exceed ±20%.
Stability
The stability of each analyte in plasma was determined by 3 QC levels in 5 replicates.
the stabilities of nifedipine in plasma samples at different concentration were examined
under different study conditions, including that the post-extracted samples in the
HPLC auto-samples at room temperature (25°C) for 0 h (fresh samples) and 7 h, the
stability of analytes in human plasma with three QC levels were stored at − 20°C
for 7 and 60 days and the stability of analytes in human plasma following two freeze-thaw
cycles and three freeze-thaw cycles. The samples were processed as described above
and the criterion for acceptability of the data is the same with that for the precision
and accuracy.
Pharmacokinetic study
20 healthy Chinese male volunteers were involved and all provided written informed
consent. Exclusion criteria included the physical examination and laboratory tests
when they were unqualified; a history of cardiovascular, hepatic, renal, psychiatric,
neurologic, hematologic, or metabolic disease; drug or alcohol abuse within 2 years
before the start of the study; allergic constitution; smoking, a history of drug allergy;
had severe low blood volume, orthostatic hypotension, arrhythmias, asthma, and a history
of glaucoma, sitting heart rate was less than 60 beats/min; consumption of any prescribed
or over-the-counter drugs within 2 weeks before the study; or participation in a similar
study within the past 6 months. The protocol was approved by the Ethics committee
of the College of Medicine, Shandong University, Jinan, China, and the study was conducted
in accordance with the declaration of Helsinki and Chinese Good Clinical Practice
guidelines.
Demographic characteristics of 20 Chinese male volunteers (mean (SD) for the overall
group included age, 25.39(1.32) years (range from 23 to 28); weight, 67.06 (8.10)
kg (rang from 56 to 80 kg); height, 1.74 (0.06) m (rang from 162 to 185); and BMI,
22.15 (1.71) (rang from 20.20 to 24.98) ([Table 2]). No volunteer was withdrawn from the pharmacokinetic study.
Table 2 The analyte response at the LLOQ in human plasma (n=5).
|
Nominal concentration (ng/mL)
|
Actual concentration (ng · mL − 1)
|
Mean
|
SD
|
RSD(%)
|
RE(%)
|
|
0.18
|
|
0.15
|
|
0.17
|
0.18
|
0.16
|
0.01
|
8.4
|
− 3.5
|
|
0.16
|
|
0.16
|
A single oral dose of either test or reference of 20 mg nifedipine sustained release
tablets with 200 mL of water were given at 7:00, and blood samples(4 mL)were collected
before and 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12, 24, and 36 h after administration
into sodium heparin (20:1) containing tubes, centrifuged at 5 000 rpm for 5 min (2 266 g)
and plasma subsequently quoted into plastic tubes and stored at − 20°C for analysis.
Drug and Statistic software (DAS 2.0, P. R. of China) was used to fit the compartmental
model of nifedipine in human and to calculate its main pharmacokinetic parameters.
Results
Method validation
Selectivity
The interference by endogenous plasma constituents with analytes and IS was assessed
by inspection of chromatograms that included typical MRM chromatograms of blank plasma,
nifedipine and IS standard, blank plasma spiked with nifedipine and IS, and plasma
from a volunteer after administration of nifedipine sustained release tablets ([Fig. 2]), retention time of nifedipine and IS were 3.65 and 2.25 min, respectively, and
no significant interferences were found at the retention times of the analytes and
IS.
Fig. 2 chromatograms of a blank plasma, b standard solution of nifedipine and IS, c blank plasma spiked with nifedipine (32.65 ng/mL) and IS, d plasma sample of subject NO. 8 after 3 h of oral administration in period 1.
Calibration curve and LLOQ
A calibration curve was established ranging from 0.17 to 102 ng/mL nifedipine in plasma.
The calibration curves were regressed using a quadratic equation with a weighting
factor of 1/x2. The coefficient of correlation of all calibration curves were more than 0.99 (n=5)
([Fig. 3]).
Fig. 3 The graph showing the coefficient of correlation of nifedipine samples (n=40) that
every calibration curves has eight concentration levels analyzed by HPLC/MS/MS.
The LLOQ was defined as the lowest concentration on the standard calibration curves
with acceptable repeatability and recovery. The analyte response at the LLOQ was should
be at least 5 times the response of blank baseline. The LLOQ was evaluated by analyzing
five replicates of spiked plasma samples at the concentration of 0.17 ng/mL for each
analyte. The precision (RSD%) and accuracy (RE%) were found to be 8.4% and − 3.5%
in [Table 2].
Recovery and matrix effect
The extraction recoveries of nifedipine were 78.05±2.32, 79.61±4.38 and 82.88±6.03
at the concentrations of 0.42, 6.53 and 81.60 ng/mL, respectively (n=5), and IS was
78.77±6.67 (101 ng/mL, n=5). The recoveries of nifedipine and IS were similar and
the data proved that the liquid-liquid extraction method was sufficient and was not
concentration-dependent. The matrix effect is a matter of notorious fact in electrospray
ionization mass spectrometry, which will influence the analyte ionization by signal
enhancement or suppression. To reduce the matrix effect, the extraction solvent and
compositions and ratio of mobile phase were investigated for finding optimal outcome.
The matrix effect was 93.06±6.02, 95.07±6.18 and 100.57±5.73 for nifedipine at the
concentrations of 0.42, 6.53 and 81.60 ng/mL, respectively (n=5), and 98.30±5.65 for
IS (n=5), suggesting that there was no significant matrix effect in this procedure
([Table 3]). These indicated that the analytical method could be kept free endogenous substances
in human plasma.
Table 3 Matrix effect and extraction recovery of nifedipine and IS in human plasma(n=5).
|
Nominal concentration (ng/mL)
|
Matrix effect
|
Extraction recovery
|
|
Mean±SD
|
RSD
|
Mean±SD
|
RSD
|
|
(%)
|
(%)
|
(%)
|
(%)
|
|
0.42
|
93.06±6.02
|
6.47
|
78.05±2.32
|
2.97
|
|
6.53
|
95.07±6.18
|
6.50
|
79.61±4.38
|
5.50
|
|
81.60
|
100.57±5.73
|
5.70
|
82.88±6.03
|
7.28
|
|
101(IS)
|
98.30±5.65
|
5.74
|
78.77±6.67
|
8.47
|
Accuracy and precision
The intra-day and inter-day precision and accuracy data for nifedipine in plasma is
summarized in [Table 4], and they were assessed by the determining of QC samples with 5 replicates for each
concentration level on the same day or on 3 consecutive days. Precision was expressed
by coefficient of variation (RSD) and accuracy by relative error (RE), and accuracy
was expressed by mean and standard deviation (SD). The assay values on both the occasions
(intra-day and inter-day) were found to be within the accepted variable limits.
Table 4 Intraday and interday precision and accuracy for analysis of nifedipine in human
plasma.
|
Nominal concentration (ng/mL)
|
intraday(n=5)
|
interday(n=15)
|
|
Mean±SD (ng/mL)
|
RSD (%)
|
RE (%)
|
Mean±SD (ng/mL)
|
RSD (%)
|
RE (%)
|
|
0.42
|
0.41±0.03
|
6.94
|
− 2.97
|
0.40±0.02
|
5.38
|
− 3.92
|
|
6.53
|
6.15±0.23
|
3.74
|
− 1.68
|
6.19±0.29
|
4.69
|
− 1.11
|
|
81.60
|
83.23±3.86
|
4.63
|
1.99
|
87.57±3.83
|
4.37
|
7.31
|
Stability studies
Stability for nifedipine after 0 h and 7 h in autosampleris were shown in [Table 5a], 2 freeze-thaw cycles and 3 freeze-thaw cycles were shown in [Table 5b], and freezed at − 20°C for 7 and 60 days were shown in [Table 5c]. The results indicated that the analytes were stable at ambient temperature for
0 h and 7 h after post extracted, at − 20°C for 7 and 60 days, and for 2 and 3 cycles
of freezing and thawing. The data conform to the acceptance criteria.
Table 5a The stability of post-extracted samples in the HPLC auto-samples at room temperature
(25°C) for 0 h and 7 h. (n=5).
|
condition
|
Nominal concentration (ng/mL)
|
Mean±SD (%)
|
RSD (%)
|
RE (%)
|
|
fresh sample(0 h)
|
0.42
|
0.43±0.03
|
7.09
|
3.87
|
|
6.53
|
7.13±0.13
|
1.78
|
11.33
|
|
81.60
|
85.94±5.51
|
6.41
|
7.42
|
|
post-extracted sample(7 h)
|
0.42
|
0.46±0.02
|
4.91
|
11.38
|
|
6.53
|
7.01±0.16
|
2.28
|
9.52
|
|
81.60
|
89.90±1.45
|
1.67
|
8.63
|
Table 5b The stability of nifedipine in human plasma following two freeze-thaw cycles and
three freeze-thaw cycles. (n=5).
|
condition
|
Nominal concentration (ng/mL)
|
Mean±SD (%)
|
RSD (%)
|
RE (%)
|
|
two freeze-thaw cycles
|
0.42
|
0.40±0.02
|
4.93
|
− 2.34
|
|
6.53
|
6.31±0.25
|
3.91
|
− 1.4
|
|
81.60
|
85.09±4.67
|
5.48
|
6.37
|
|
three freeze-thaw cycles
|
0.42
|
0.42±0.03
|
6.13
|
0.48
|
|
6.53
|
6.44±0.37
|
5.73
|
− 1.44
|
|
81.60
|
82.65±2.76
|
3.33
|
1.29
|
Table 5c The stability of nifedipine in human plasma with three QC levels at − 20°C for 7
and 60 days (n=5).
|
condition
|
Nominal concentration (ng/mL)
|
Mean±SD (%)
|
RSD (%)
|
RE (%)
|
|
− 20°C, 7days
|
0.42
|
0.38±0.01
|
2.97
|
− 7.81
|
|
6.53
|
6.51±0.27
|
4.15
|
1.64
|
|
81.60
|
78.41±7.42
|
9.46
|
− 1.99
|
|
− 20°C, 60days
|
0.42
|
0.37±0.03
|
8.72
|
− 9.86
|
|
6.53
|
6.50±0.52
|
8.02
|
1.49
|
|
81.60
|
79.04±2.64
|
3.34
|
− 1.2
|
Clinical application
The validated method was applied to a pharmacokinetic study for determination of nifedipine
concentration in human plasma after a single oral dose of its sustained release tablets.
A 1/C weighted coefficients regression analysis and 2-compartmental model were used
to fit the disposition of nifedipine in Chinese volunteers. The mean concentration-time
curve is shown in [Fig. 4]. The main pharmacokinetic parameters are shown in [Table 6]. The results were found to be within the assay variability limits during the entire
process.
Fig. 4 Mean plasma concentration-time curve of nifedipine after oral administration of 20 mg
nifedipine sustained-release tablet (n=20).
Table 6 Main pharmacokinetic parameters of nifedipine after administration of 20 mg nifedipine
sustained-release tablets (n=20, Mean±SD).
|
Parameter
|
Value
|
|
AUC(0–36)(ng · h/mL)
|
546.49±162.28
|
|
AUC(0-∞)(ng · h/mL)
|
564.05±176.74
|
|
MRT(0–36) (h)
|
8.40±1.60
|
|
MRT(0-∞) (h)
|
9.42±2.66
|
|
CL (L/h/kg)
|
0.03±0.01
|
|
CLz (L/h/kg)
|
0.04±0.02
|
|
Vz (L/kg)
|
0.37±0.17
|
|
Cmax (ng/mL)
|
76.69±19.51
|
|
Tmax (h)
|
2.80±0.50
|
|
t1/2α(h)
|
6.78±2.52
|
|
t1/2β(h)
|
6.82±2.53
|
|
t1/2z(h)
|
6.69±2.22
|
|
K10(1/h)
|
0.11±0.04
|
|
K12(1/h)
|
0.03±0.04
|
|
K21(1/h)
|
0.12±0.04
|
|
VRT(0–36) (h2)
|
55.22±18.42
|
|
VRT(0-∞) (h2)
|
96.21±72.60
|
Discussion
Mass spectrometry optimization
In order to optimize ESI conditions for nifedipine and IS, both the positive and negative
ion modes were investigated. However, a poor linearity in positive ionization condition
and a good response was found in negative ionization mode. The solutions containing
nifedipine and IS were injected directly into the mass spectrometer. Under these conditions,
the analytes yielded major ions at m/z 345.1 for nifedipine and m/z 150.1 for IS.
Each of precursor ions was subjected to collision-induced dissociation to determine
the resulting product ions. The full-scan negative product ion mass spectra of precursor
ion spectrum of nifedipine and IS and the product ion mass spectra of nifedipine and
IS are shown in [Fig. 5]. The results showed that the most sensitive and abundant mass transitions were m/z
345.1→222.2 for nifedipine and 150.1→107.1 for IS. The MRM state file parameters were
the optimized values for the sensitivity and specificity required for nifedipine.
Fig. 5 full-scan negative product ion mass spectra of a precursor ion spectrum of nifedipine; b product ion spectrum of nifedipine; c precursor ion spectrum of IS; d product ion spectrum of IS.
Selection of IS
It was difficult to find a compound that could ideally mirror the analyte to serve
as a suitable IS that should mimic the analyte during extraction and have a stationary
response, especially, for HPLC-MS/MS, matrix effect used to induce poor analytical.
Several compounds were investigated, such as nitrendipine, diazepam, hydrochlorothiazide,
glycyrrhetinic acid and acetaminophen. Nitrendipine had long retention time, diazepam
was no peak under the negative ion detection mode, hydrochlorothiazide had serious
smear under the conditions used and glycyrrhetinic did not proper product ion under
product scan mode. None of these, but finally acetaminophen ([Fig. 1b]) was found to be the most appropriate for the present purpose. The retention time
of acetaminophen was short to that of nifedipine. Chromatograms were obtained and
no significant direct interference in the MRM channels at the relevant retention time
was observed and it could save time for a large samples analysis.
Sample pre-treatment
Protein precipitation, Liquid-liquid extraction (LLE) and solid phase extraction (SPE)
were tested and compared, with acetonitrile, toluene, and ether-n-hexane (3:1, v/v)
as protein precipitation solvents, but protein precipitation was easy to dilute the
sample and failed to sufficiently remove endogenous interference. SPE had too many
disturbances to good reproducibility and recovery [16]
[19]. What is more, the column for SPE is expensive and not suitable for high-throughput
analysis when a large number of samples were processed. LLE was used for producing
chromatographia clean samples in the study, which contributed to minimizing ion suppression
and matrix effects in HPLC-MS/MS. LLE with various extraction solvents, including
diethyl ether, chloroform, dichloromethane and ethyl acetate, were investigated and
evaluated for acceptable extraction recoveries and matrix effect. Diethyl ether, extraction
recoveries were about 42.12% and unacceptable, chloroform was easy to emulsify when
the sample was made vortex and mixed and its recover was about 54.20%, dichloromethane,
matrix effect were about 75.23% and disturb the results. Ethyl acetate was with several
obviously advantages, firstly, the upper organic phase was transferred easily, secondly,
ethyl acetate possesses less toxicity, good stability and repeatability, finally,
extraction recoveries were approximately 81.63% ([Table 7]). Furthermore, it was also tested whether sodium hydroxide (0.05 mol/L) was added
to extraction solvents, and there were not obvious difference for peak area and retention
time.
Table 7 The development summary of liquid-liquid extraction procedure (20 ng/mL, n=5).
|
extraction solvent
|
Matrix effect
|
Extraction recovery
|
|
Mean±SD (%)
|
RSD (%)
|
Mean±SD (%)
|
RSD (%)
|
|
diethyl ether
|
71.53±9.63
|
11.32
|
42.12±5.76
|
19.77
|
|
chloroform
|
68.11±2.11
|
10.97
|
54.20±6.89
|
8.15
|
|
dichloromethane
|
75.23±5.22
|
14.33
|
68.42±8.71
|
17.88
|
|
ethyl acetate
|
97.56±5.12
|
5.40
|
81.63±5.10
|
4.32
|
Solvents included methanol, acetonitrile, 5mmol/L ammonium acetate and mobile phase
for reconstituting residues were also investigated to optimize the chromatographic
behaviors for optimizing peak shape and minimum peak response. Mobile phase with good
peak shape and minimum response was adopted.
Liquid chromatography
A simple chromatographic separation was developed for acquisition of good separation
with a short run time. The feasibility of various mixtures of solvents such as acetonitrile
and methanol using different water phase, including 5 mM ammonium acetate (pH=6.62),
2 mM ammonium acetate (pH=7.19), water and 1% methanoic acid were tested, along with
altered flow-rates (in the range of 0.5–1 mL/min), was tested to identify the optimal
mobile phase that produced the best sensitivity, efficiency and peak shape. It was
found that acetonitrile mixed with 5 mM ammonium acetate solution (pH=6.62) (60:40,
v/v) could achieve this purpose in negative ionization mode and finally used as the
mobile phase. A flow rate of 0.8 mL/min permitted a run time of 5 min.
Conclusions
In summary, a highly sensitive, specific, reproducible and high-throughput HPLC-MS/MS
method that we developed and validated based on the procedure of LLE for determination
of nifedipine with IS. The procedure was fully validated to meet the requirements
for sensitivity, accuracy and precision from State Food and Drug Administration and
GLP Guidelines for industry. According to the validation parameters, the developed
method could be useful for nifedipine pharmacokinetic studies and routine therapeutic
drug monitoring with desired precision and accuracy.
