INTRODUCTION:
Carisoprodol
(Fig 1)is a muscle relaxant, acts by affecting the
central nervous system making it popular drug of abuse1,2. It is
possible that carisoprodol induced muscle relaxation
results from a slight depression of all neurons at synaptic junctions within
the central nervous system3. After oral administration, it gets
metabolized to meprobamate, an anti-anxiety agent
produces sedation via GABAA receptors, prescribed primarily to treat
anxiety, tension and associated muscle spasms4.
Fig 1:Chemical
Structure of Carisoprodol
There
were few LC-MS/MS methods developed for estimation of Carisoprodol in tablet
dosage forms5 and for simultaneous estimation of Carisoprodol
with other drugs6-8 and metabolites9,10
in biological fluids. But there were no methods developed for the estimation of
Carisoprodol alone in human plasma which is very
useful for bioavailability and bioequivalence studies.
The
present study describes a simple, rapid and sensitive liquid chromatography
with electro spray ionization-tandem mass spectrometric method for the quantization
of Carisopridol in human plasma using Carisoprodol Methyl D3 as IS with a
chromatographic run time of 2.0 min.
MATERIALS AND
METHODS:
Standards and Chemicals:
The
reference sample of Carisoprodol (98.95%) and IS of Carisoprodol Methyl D3 (98%) were obtained from Clearsynth Labs Limited (Mumbai, India). Water used for the
LC-MS/MS analysis was prepared by Milli Q Water
Purification System processed from Millipore (Bangalore, India). HPLC grade
methanol, acetonitrile and analytical grade ammonium format
were purchased from Merck Ltd (Mumbai, India). The control K2 human
plasma sample was procured from Deccan’s Pathological Labs (Hyderabad, India).
LC-MS/MS Instrument and Conditions:
A
Shimadzu HPLC system (Model SIL-HTC) equipped with a Zorbax
XDB (Phenyl) column (75mm X 4.6 mm, 3.5µm), a binary LC-20AD prominence pump,
an autosampler and a solvent degasser was used for
the study. An aliquot of 15µl of processed sample was injected into the column,
which was at ambient (20±5şC) temperature. An isocratic mobile phase composed
of a mixture of 10mM ammonium format and acetonitrile
(15:85,v/v) was used at a flow rate of 1.2ml/min.
Quantification
was achieved with MS-MS detection in positive ion mode for the analyte and the IS using Bios stems mass spectrometer
(Model: MDS Sciex API 3000). The ion spray voltage
was set at 5500V. The source parameters viz. the nebulizer gas(NED),
Curtain gas (CUR) and Collision gas (CAD) were set at 8,8 and 6psi
respectively. The compound parameters viz. the declustering
potential (DP), collisional energy (CE), entrance
potential (EP), collisional cell exit potential (CXP)
and focusing potential 40, 12, 10, 9 and 350V for Carisoprodol
and 50, 12,10,9 and 350V for the IS. Detection of the ions was carried out in
the Selected Ion Monitoring (SIM) mode by monitoring the transition pairs of
the m/z 261.3 precursor ion to the m/z 176.1 product
ion for Carisoprodol and m/z 264.4 precursor ion to the
m/z 179.2 product ion for the IS. Quadrupoles (Q1
and Q3) were set at unit resolution.
Preparation of Stock and Working
Solutions:
Two standard stock solutions of Carisoprodol
were prepared separately in HPLC grade methanol (1mg/ml). Their concentrations
were corrected according to the actual amount weighed accounting for its
potency. Working standard solutions necessary for plotting the the calibration curve
(CC) samples were prepared by appropriate dilution of the one of the above
stock solution of the Carisoprodol using a mixture of
methanol and water (50:50, v/v; diluent). Quality control (QC) samples for
determination of accuracy and precision were prepared by appropriate dilution
of the second standard solution prepared above using the same diluent. The concentration of the QC samples were selected from the five
different levels of the calibration curve range. A 1mg/ml of Carisoprodol Methyl D3 stock solution was
prepared by dissolving the compound in HPLC grade methanol. The working
concentration of Carisoprodol Methyl D3
(15ng/ml) was prepared from the above stock solution using the diluent.
Preparation
of Calibration Curve Standards and Quality Control Samples in Human Plasma:
Six slots of K2 EDTA human plasma were
screened and used to prepare calibration curve standards and QC samples. After
bulk spiking, aliqouts of 200µl for CCs and 200µl for
QCs of spiked plasma samples were pipetted into prelalbelled microcentrifuge
tubes (2ml) and then all the bulk spiked samples were stored in a deep freezer
at -70±10şC. Calibration samples were prepared by spiking 475µl of control K2
EDTA human plasma with the 25µl of working standard solution of the analyte as a bulk, to obtain Carisoprodol
concentration levels of 25, 50, 150, 300, 600, 1200, 1800, 2400 an 3000ng/ml as
a single batch at each concentration. Similarly QC samples were also prepared
as a bulk based on an independent weighing of the standard drug, at
concentrations of 25.20 (Lower limit of quantization quality control, LLOQ QC),
75.21 (low quality control, LQC), 450.36(Medium quality control, MQC 1),
1501.20 (MQC 2) and 2700ng/ml (High Quality Control, HQC) as a single batch at
each concentration.
Sample
Preparation Protocol:
All frozen subject samples, calibration standard and
QC samples were thawed and allowed to equilibrate at room temperature prior to
analysis. The samples were vortexed to mix for 10
seconds prior to spiking. A 250µl of aliquot of human plasma sample was mixed
with 25µl of IS working solution (15µg/ml). To this 250µl of 10mM ammonium format
buffer was added and vortexed. After vortex mixing,
5ml of extraction solvent (ethyl acetate:n-hexane
80:20) was added, shaken for 20min on reciprocating shaker at 200rpm. Samples
were centrifuged at 4000rpm for 10min at 4şC. Supernatant organic layer (4.0ml)
was transferred to prelabelled dry test tubes and
evaporated to dryness at 40şC under a gentle stream of nitrogen. The residue
was reconstituted with 1000µl of mobile phase and 15µl were injected into
LC-MS/MS system.
Method
Validation Parameters:
The validation of the above method was carried out
according to the US FDA guidelines11. A carryover experiment was performed to verify any
carryover of the analyte and the IS, which may
reflect in the subsequent runs. The carryover test samples were injected in the
following sequence i.e blank plasma sample →
six samples of LLOQ → blank plasma sample → Upper Limit of
Quantification (ULOQ) sample → blank plasma sample to check the carryover
effect. The sensitivity of the method was assessed in eight different sources
of plasma of which six were normal K2 EDTA human plasma and one each
of lipemic and hemolyzed
plasma. Sensitivity of the method was assessed by analyzing six sets of spiked
plasma samples at the lowest level of the calibration curve concentration
(LLOQ). The matrix effect, expressed as the IS normalized matrix factor (MF)
was assessed by comparing the mean area response of post extraction spiked
samples with a mean area of aqueous samples (neat samples) prepared in mobile phase solutions at LQC and
HQC levels. The overall precision of the matrix factor was expressed as the
coefficient of variation (CV).
Peak response area
ratio in presence of matrix ions
Matrix Factor =
Peak response area
ratio in absence of matrix ions
The linearity of the method was determined by analysis
of standard solutions containing nine nonzero concentrations. Intraday accuracy
and precision were determined using six replicates of LLOQ QC, LQC, MQC 1, MQC
2 and HQC samples in a single day. Interday accuracy and precision were
assessed by analyzing six batches of samples on three consecutive days. The
precision (% CV) at each concentration level from the nominal concentrations
should not be greater than 15% except for LLOQ QC where it should be 20%. The
accuracy (%) must be within ±15% of their nominal value at each QC level except
LLOQ QC where it must be within ±20%. Recovery for the analyte
and the IS was calculated by comparing the mean detector response of six sets
of pre-extraction spiked samples (Spiked before extraction) to that of six sets
of neat samples (aqueous) at each concentration level. Recovery of Carisoprodol was determined at a concentration of 75.21
(LQC), 1501.20 (MQC 2) and 2700ng/ml (HQC) where as for the IS was determined
at a concentration of 375ng/ml.
RESULTS AND DISCUSSION:
The current method was developed using an electro
spray ionization source in the positive ionization mode. The protonated form of the analyte
and the IS, [M+H]+ ion was the precursor ion in the Q1 spectrum
and was used as the precursor ion to obtain Q3 product ion spectra.
The most sensitive mass transition was observed from m/z 261.3 to 176.1 for Carisoprodol and from m/z 264.4 to 179.2 for the IS. The
most intense and consistent product ion Q3 MS spectra of the analyte and the IS were obtained by optimizing the
collision energy and collision cell exit potential. The source parameters like
nebulizer gas (NED), collision gas (CAD), temperature and ion spray voltage
were optimized to obtain adequate and reproducible response for the analyte. The LC-SIM technique was chosen for the assay
development due to its inherent selectivity and sensitivity.
The method developed includes mobile phase selection,
column type, flow rate and injection volume. Acetonitrile
and methanol were tried in different ratios with buffers like ammonium acetate,
ammonium formate as well as acid additives like
formic acid and acetic acid in varying strengths. It was observed that 10mM Ammonium
formate and acetonitrole
(15:85, v/v) as the mobile phase was the most appropriate to give the best
sensitivity, efficiency and peak shape. The use of short chromatography column Zorbax XDB Phenyl (75 X 4.6 mm, 3.5µm) helped in the
separation and elution of analyte and the IS in a
very short time. The total chromatographic run time was 2.0min for each run. Bological samples are complex and contain many endogenous
components. To develop a sensitive analytical method for biological samples one
should have a proper extraction technique which can produce good recovery with
minimal or no matrix effect. Liquid Liquid Extraction
(LLE) was carried out using ethyl acetate and n-hexane. The supernatant was
evaporated and the residue was reconstituted with the mobile phase. An ideal
internal standard sould mimic the analyte
during ionization, separation and extraction. Stable isotope labelled drugs or deuterated
compounds are preferred internal standards for the LC-MS/MS analysis. Carisoprodol Methyl D3 which is the deuterated compound was selected as IS.
Carryover
test:
Carryover test is performed to ensure that it does not
affect the accuracy and precision of the proposed method. No significant
carryover was observed in the blank sample when injected after the injection of
highest concentration of the analyte along with the
working concentration of the IS (ULOQ), which indicates no carryover of the analyte and the IS in subsequent samples.
Selectivity:
The method selectivity was evaluated by injecting the
blank human plasma extract and an extract spiked only with the IS. No
significant direct interference in the blank plasma traces was observed from
endogenous components at the retention time of the analyte
and the IS, as shown in Fig 2, 3 and 4.
Sensitivity
LLOQ is the lowest limit of reliable quantification
for the analyte and was set at 25ng/ml. Signal to
Noise ratio was measured at this concentration and was found to be ≥10.
The precision and accuracy at LLOQ concentration were found to be 6.07% and
101.89% respectively.