Bio-analytical chiral chromatography method for the enantioselective separation of carbinoxamine maleate in human plasma
© Tadiboyina et al. 2015
Received: 1 June 2015
Accepted: 3 November 2015
Published: 10 November 2015
A selective chiral high-performance liquid chromatography (HPLC) method was developed and validated to separate and quantify the (d) and (l) carbinoxamine enantiomers in human plasma.
Plasma samples were extracted by liquid-liquid extraction. The separation of carbinoxamine enantiomers and internal standard (IS, pargeverine hydrochloride) was achieved on an amylose tris(5-chloro-2-methylphenylcarbamate) column with a mobile phase of n-Hexane/isopropanol/ethanol/diethyl amine (850:75:75:0.1, v/v/v/v) at a flow rate of 0.8 mL/min. The ultraviolet (UV) detection wavelength was set at 220 nm.
Baseline separation of carbinoxamine enantiomers and IS, free from endogenous interferences, was achieved in less than 15 min. Ratio of peak area of each enantiomer to IS was used for quantification of plasma samples. Linear calibration curves were obtained over the range of 20–7500 g/mL in plasma for both enantiomers (R 2 > 0.99). The mean extraction recoveries were 103.8 ± 1.5 and 94.5 ± 1.8 % for (d) and (l) enantiomers of carbinoxamine enantiomers and 96.35 % for IS from human plasma. The mean relative error (RE %) of accuracy and the mean relative standard deviation (RSD %) of intra-day and inter-day precision for both enantiomers were <10 %.
The method was validated with accuracy, precision, recovery, and stability and can be used to determine the pharmacokinetics of carbinoxamine enantiomers in human plasma.
Methods for the determination of CA in biological fluids and dosage forms
BDS Hypersil C8 (100 × 4.6) column; mobile phase: acetonitrile:buffer (80:20 v/v); buffer (25 mM ammonium formate)
MRM mode, electrospray-positive ionization
(GeetaBhavani et al. 2014)
Waters PVA-Sil HPLC (50 × 4.0) mm column; acetonitrile:ethylacetate:water:methanol: formic acid:morpholine (500:200:100:60:0.2:0.025 v/v/v/v)
Sciex API 4000, triple quadrupole mass spectrometer
(Michael et al. 2008)
Ion exchange chromatography
Absorbance at 264 mμ
Pharmaceutical formulations (syrups, drops, and tablets)
(Ramadan and Mandil 2006)
Spectrophotometry and HPLC
ACE C18 (250 × 4.6 mm) column; mobile phase: gradient elution program
DAD detection, absorbance at 210–300 nm range
Pharmaceutical formulations (capsule)
(Ismail and Feyyaz 2012)
Acetonitrile, 0.01 sodium perchlorate (pH 3)
DPP and DCP
Mobile phase: phosphate buffer (pH 1.69) using dropping mercury electrode (DME) vs. Ag/Agcl
Diffusion currents (id) and peak currents (ip)
Pharmaceutical formulations (tablets, capsules, syrups, and oral drops)
(Abdul et al. 2009)
Ion pair reverse phase high-performance liquid chromatography
C18 (300 × 3.9 mm); mobile phase: methanol:monobasic phosphate buffer (60:40 v/v) with 1 ml phosphoric acid, 0.5 ml TEA and 0.25 g sodium lauryl sulfate
UV detection 300 nm
Pharmaceutical formulations (tablets)
(de Carina and Marcone 2009)
Perchloric acid titration
Titrate: 0.1 N perchloric acid with crystal violet TS as indicator
Blue-green end point
Capillary zone electrophoresis
Pre-column dervetization using sulfated beta-cyclodextrin as a chiral selector; mobile phase: tris buffer (100 mM pH 4.6)
UV detection 200 nm (anode at detection side)
(Yi-Fen et al. 2000)
Amylose-based chiral column (250 × 4.6 mm); mobile phase: hexane:ethanol (90:10 v/v)
UV detection 220 nm (22 °C)
Chemicals and materials
Carbinoxamine maleate and pargeverine hydrochloride standards were gifted by RL Fine Chemicals, Bengaluru, India. n-Hexane, 2-proponol, ethanol, and diethylamine were procured from Merck, Mumbai, India. High-performance liquid chromatography (HPLC)-grade water was obtained from a Milli-Q unit (Millipore, Milford, USA). Blank human plasma was obtained from JSS Medical College and Hospital, JSS University, Mysuru, India.
The instrumentation consisted of a Shimadzu Prominence LC-20 AD ultrafast liquid chromatography (UFLC) equipped with a 1260 binary pump VL (35 MPa), Prominence SIL-20ACHT Auto sampler, and Prominence SPD-M20A Diode array detector. All weighings for analysis were performed on a Shimadzu electronic analytical balance AY-220 (Shimadzu). Plasma samples were sonicated on a Mark ultrasonic sonicator and vertexed on a Remi cyclomixer; centrifugation was done using REMI centrifuge model number 412 LAG (REMI Instruments Division, Vasai, India). Water used for analysis was prepared from Millipak Express 20 filter unit. Microsoft Excel 2007 was used for analysis of validation results.
The chromatographic separations were carried out on amylose tris(5-chloro-2-methylphenylcarbamate) column. The mobile phase was a mixture of n-Hexane/isopropanolthanol/diethyl amine (850:75:75:0.1, v/v/v/v) with 0.8 mL/min of flow rate, and detection was performed by a photodiode array detector (PDA) at 220 nm.
Preparation of stock and standard solutions
Stock solutions of CA and PGV in isopropanol were prepared separately at a concentration of 10 μg/mL. A series of standard mixture solutions were prepared by the appropriate dilution of the stock standard solutions with isopropanol to a concentration range of 0.4–1000 μg/mL for CA and 40 μg/mL for PGV.
Preparation of samples
The plasma samples were thawed at room temperature. The samples were vortexed adequately before pipetting. To a 200-μL aliquot of plasma, 100 μL of racemic CA stock solution (0.4–1000 μg/mL), 100 μL of IS solution (40 μg/mL), and 100 μL of 1 M NaOH were added and vortexed for 1 min in a 2-mL Eppendorf tube. The mixed samples were then extracted with 1.5 mL of dichloromethane: n-Hexane (1:2), by vortex mixing for 2 min. After centrifugation at 5000 rpm for 15 min, 1.0 mL of the upper organic layer was transferred to another tube. Extracts were concentrated to dryness at the 40 °C under a gentle stream of nitrogen and reconstituted with 150 μL of ethanol. A 25-μL aliquot of the solution was injected into the UFLC system for the analysis.
Bio-analytical method validation
The plasma calibration curve was constructed using a blank sample (matrix sample processed without analyte or internal standard), a zero sample (matrix sample processed without analyte but with internal standard), and six non-zero samples (matrix samples processed with analyte and internal standard) covering the expected range including lower limit of quantification (LLOQ), 20–7500 ng/mL with 2000 ng/mL of IS concentration.
The efficiency of carbinoxamine enantiomers and IS extraction from human plasma was determined by comparing the responses of the analyte extracted from six reproduce QC samples with the response of analyte from neat standards at equivalent concentrations by a liquid-liquid extraction process. Recoveries of CA were determined at the LLOQ, QC low, QC medium, and QC high concentrations that is, 20, 300, 1200, and 5000 ng/mL. Whereas the recovery of the IS was determined at a single concentration of 2000 ng/mL.
Accuracy and precision
The intra-assay accuracy and precision were estimated by analyzing six replicates containing CA at four different QC levels, that is, 20, 300, 1200, and 5000 ng/mL. The inter-assay precision was estimated by studying the quality control samples on four different runs. The criteria for acceptability of accuracy data within 85–115 % of the actual values and ±15 % relative standard deviation (RSD) except for LLOQ for precision.
Stability tests were conducted to evaluate the stability of CA enantiomers in plasma samples under different conditions. In-injector stability (24 h), bench-top stability (12 h), freeze-thaw stability (3 cycles), and freezer stability (80 ± 10° C for 25 days) were tested at LQC (300 ng/mL) and HQC (5000 ng/mL) levels using six replicates at each level. Samples were said to be stable if assay values were within the acceptable limits (i.e., 85–115 % accuracy and ±15 % RSD from fresh samples).
Results and discussion
Method development and optimization
Selection of chiral stationary phase
The finest chiral stationary phases (CSP) for the separation of aromatic compounds with functional groups such as carbonyl, alcohol, and amine (like carbinoxamine) are amylose-based polysaccharide stationary phases (LCGC 2008). Immobilized polysaccharide columns are compatible with a wide range of solvents; hence, they can be operated in both reverse phase and normal phase which increased the range of applications (Othman et al. 2012). Hence, the coated amylose-based polysaccharide column was selected for screening.
The CSP is amylose tris(5-chloro-2-methylphenylcarbamate) bonded to silica gel. The separation of enantiomers may be attributed to hydrogen bonding interactions between the amine group of solute and the hydrophilic carbamate group on the CSP. Steadying effect on the solute-CSP complex will be there for the solutes having aromatic functional groups (Irving and Rose 1987). This type of steadying effect (or) stabilization effect may also exist in CA owing to the presence of aromaticity.
Selection and optimization of additives
As polysaccharide-based CSPs generally show a higher success rate in resolving enantiomers under normal-phase elution (Brian 2010); hence, this mode was explored first. The selection of mobile phases was initiated with traditional alkane/alcohol mixtures (Chiral Technologies 2004). n-Hexane was given first preference as it is comparatively green (Paul et al. 2012). For separation of most of the analytes containing basic and/or acidic functional groups, the additive plays a major role in increasing the chromatographic efficiency (Zhang et al. 2012). The additives were used in the present word in order to improve the peak shape. All chromatographic parameters were kept constant (flow rate, 0.8 mL/min, ambient column oven temperature; injection volume, 25 μL; wavelength, 220 nm) except the % additive added. Diethylamine and triethylamine were often used as a modifier for basic drugs with amine groups to ensure elution from the column and to obtain good peak shape (Toussaint et al. 2000). Hence 0.01 % diethylamine was introduced into the mobile phase, CA enantiomers were eluted at around 10 min, and the peaks were highly asymmetric. Further increase in diethylamine content (0.1 %) has significant impact on the peak shape, whereas the mobile phase without diethylamine significantly distorted the peak shape.
Selection of alcoholic modifiers
Screening of alcoholic modifiers
Alcoholic modifier (×) per 100 mL of mobile phase (mL)
Poor resolution between the enantiomers, tailing of peaks observed
Enantiomers eluted very lately and the peak shape is broad.
Enantiomers eluted early but not separated completely; no resolution between the peaks.
No proper separation of enantiomers
Enantiomers eluted early with good resolution and capacity factor.
System suitability parameters
Theoretical plates (N)
Tailing factor (T)
Capacity factor (K)
Pargeverine HCl (IS)
Optical rotation of CA enantiomers
The bio-analytical validation was performed according to FDA guidelines (FDA guideline 2001).
The calibration standard curve had a reliable reproducibility over the standard concentrations across the calibration range. The calibration curve was constructed by determining the best fit of peak area ratios (peak area analyte/peak area IS) vs. concentration and fitted to the y = mx + c. The average regression (n = 3) was found to be ≥0.996 for both the (d) and (l) enantiomers of CA. The lowest concentration with the RSD <20 % was taken as the LLOQ and was found to be 20 ng/mL.
Recovery of Carbinoxamine enantiomers (n = 6)
Mean recovery % ± SD
Quality control sample (ng/mL)
LQC ± 300
103.1 ± 2
101.7 ± 7.60
MQC ± 1200
94.4 ± 3
104.5 ± 2.7
HQC ± 5000
97.4 ± 6
94.1 ± 8.9
Accuracy and precision
Intra- and inter-day precision of determination of CA in human plasma
Theoretical concentration (ng/mL)
Measured concentration (ng/mL), (n = 6)
Mean ± SD
20.1 ± 0.85
19.4 ± 0.35
320 ± 5.28
307 ± 6.35
1228 ± 10.4
1261 ± 9.37
4734 ± 24.5
4675 ± 20.15
20.5 ± 0.17
20.6 ± 0.74
314 ± 2.38
324 ± 7.12
1274 ± 11.3
1257 ± 18.74
4735 ± 70.1
4823 ± 88.45
Stability data CA quality controls in human plasma
Nominal con. (ng/mL)
Mean ± SD
Precision (% CV)
317 ± 9.07
286 ± 8.34
12 h (bench-top)
323 ± 5.01
257 ± 2.76
24 h (in-injector)
326 ± 9.72
317 ± 5.2
340 ± 12
296 ± 10
25 days at −80 °C
338 ± 1.47
318 ± 3.4
4681 ± 54.3
4478 ± 28.9
12 h (bench-top)
4734 ± 47.4
5183 ± 38.5
24 h (in-injector)
4697 ± 35.1
5013 ± 60.7
4737 ± 70.2
4826 ± 51.2
25 days at −80 °C
4705 ± 35.4
4478 ± 32.7
The developed method was found to be sensitive, specific, and robust for quantification of both the (d) and (l) enantiomers of carbinoxamine in human plasma. The method involved sample preparation with adequate recovery by liquid-liquid extraction. Method parameters like % alcoholic modifier, % basic additive, and flow rate were successfully adjusted. The method ensured as a robust one from the responses obtained. Even for injection volume 25 μL the method is quite sensitive. The method is user-friendly because of using green solvents n-Hexane and ethanol. It is concluded that the method is suitable for the routine quantification of (d) and (l) enantiomers of CA in human plasma.
Authors are thankful to The Principal, JSS College of Pharmacy, JSS University, Mysuru, India for providing the necessary facilities. The authors express gratitude to Dr. Ramesh A. R, Vice President, R L Fine Chem, Bengaluru, for providing the racemic carbinoxamine maleate drug as a gift sample. Authors are beholden to University Grants Commission, New Delhi for the financial assistance (Ref: F.No.40-271/2011 (SR) dated 10.01.2013).
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