Determination Of Enantiomeric Impurities Research Articles

Determination of Enantiomeric Impurity in Tegoprazan Drug Substance by HPLC

ABSTRACTTegoprazan is a novel potassium‐competitive acid blocker used for gastroesophageal reflux disease. It is administered as the (S)‐ and (R)‐enantiomers are considered impurities that should be controlled. The objective of this work was to establish a chiral analytical method for the determination of the enantiomeric impurity of tegoprazan in the drug substance by high‐performance liquid chromatography (HPLC). Enantioselective separation was performed on a Chiralpak alpha1‐acid glycoprotein (AGP) column (4.6 mm × 250 mm, 5.0 µm) at 30°C utilizing a mobile phase consisting of 5 mmol L−1 ammonium acetate buffer (pH 6.0) and 2‐propanol (93:7, v/v) at a flow rate of 0.7 mL min−1 with a detection wavelength of 220 nm. The effects of chromatographic conditions, including the type and concentration of organic modifiers, buffer pH, buffer concentration, flow rate, and column temperature, were thoroughly investigated. Under the optimized conditions, the resolution between (S)‐ and (R)‐tegoprazan was greater than 3.0. Moreover, the developed method was validated in terms of linearity, limits of detection (LOD) and quantitation (LOQ), accuracy, precision, and stability. Each enantiomer showed a good linear relationship over the concentration range of 1.0–100 µg mL−1, with the correlation coefficient of 0.9999. The LOD and LOQ were 0.1 and 0.3 µg mL−1, respectively. In addition, the recoveries ranged from 94.10% to 99.39% with a relative standard deviation (RSD) of 1.9%. Application of this proposed method to determine the enantiomeric impurity in tegoprazan drug substance proved satisfactory. The quantitative results were about 2.0%, demonstrating that enantiomeric impurity remained in the tegoprazan drug substance and should be controlled in the quality criteria.

Development and validation of a sensitive and robust chiral method for (R)-propylene oxide: From chiral gas chromatography to chiral liquid chromatography with pre-column derivatization

Propylene oxide (PO) is an important chemical commodity and a chiral building block to prepare pharmaceutical compounds. It is crucial to develop a sensitive and robust chiral method that can be used to assess the enantiomeric purity of PO and safeguard the quality and safety of pharmaceutical products in commercial quality control (QC) laboratories. A chiral gas chromatography (GC) method based on direct enantioseparation of PO enantiomers on a 50-meter Chiraldex™ A-TA open capillary column was successfully developed for determination of enantiomeric impurity (S)-PO in (R)-PO. However, it was revealed that the chiral GC method lacked adequate robustness for routine use. Consequently, a chiral high-performance liquid chromatography (HPLC) method was developed through pre-column derivatization of PO with 2-naphthalenethiol followed by enantioseparation on a Lux Cellulose-1 (250 mm × 4.6 mm, 5 µm) column. The chiral HPLC method has been demonstrated excellent specificity, sensitivity, accuracy and robustness through method validation and robustness evaluation, thus establishing its suitability for routine use in QC laboratories.

A specific chiral HPLC method for lifitegrast and determination of enantiomeric impurity in drug substance, ophthalmic product and stressed samples

Lifitegrast is a lymphocyte function-associated antigen-1 (LFA-1) antagonist used to treat the indications and symptoms associated with dry eye disease (DED), one of the most common ocular surface diseases. Lifitegrast has a chiral center, and the S-enantiomer (S-Lif) is responsible for the therapeutic effects, while the R-enantiomer (R-Lif) lacks efficacy in the treatment of DED. Lifitegrast ophthalmic solution containing 5% lifitegrast was approved by the United States Food and Drug Administration (FDA) in July 2016 for the treatment of DED in patients 17 years of age and older. The objective of this study was to develop a chiral HPLC method for the determination of the enantiomeric impurity of lifitegrast in the drug substance and in the ophthalmic product. In addition, we aimed to investigate the effect of stress and stability conditions on the enantiomeric purity of lifitegrast in both drug substance and ophthalmic solution. During the method development studies, four known lifitegrast impurities (Lif. Imp. A-D) and stressed lifitegrast samples were injected to ensure the specificity of the developed method. The enantiomers of lifitegrast are well separated with a resolution of higher than 4.0. They are also well separated from the peaks of the diluent, impurities, and the placebo used to prepare the ophthalmic solution without interference in 20 min. Chiral separation was achieved using a Chiralpak AD-H column (250 × 4.6 mm, 5.0 μm) at 40 °C with a mobile phase consisting of a mixture of n-hexane, 2-propanol, and formic acid (500:500:2, v/v/v) at a flow rate of 1.0 mL/min and a detection wavelength of 260 nm. Methanol was used as the diluent, and the drug substance solution was found to be stable for 48 h at 15 °C. The optimized chiral HPLC method for lifitegrast was validated according to ICH Q2, and the calibration curves showed excellent linearity for R-Lif (0.0369 – 1.816 µg/mL). This is the first stability-indicating, specific / selective, sensitive, linear, precise, accurate, and robust chiral HPLC method for the determination of R-Lif in S-Lif. The amount of enantiomeric impurity R-Lif in S-Lif increased under all stress and photostability test conditions without exceeding the acceptable impurity limit, with the most significant increase observed at elevated temperatures (105 °C) for both the drug substance in powder form and the ophthalmic drug solution.

Chiral Monolithic Silica-Based HPLC Columns for Enantiomeric Separation and Determination: Functionalization of Chiral Selector and Recognition of Selector-Selectand Interaction.

This review draws attention to the use of chiral monolithic silica HPLC columns for the enantiomeric separation and determination of chiral compounds. Properties and advantages of monolithic silica HPLC columns are also highlighted in comparison to conventional particle-packed, fused-core, and sub-2-µm HPLC columns. Nano-LC capillary monolithic silica columns as well as polymeric-based and hybrid-based monolithic columns are also demonstrated to show good enantioresolution abilities. Methods for introducing the chiral selector into the monolithic silica column in the form of mobile phase additive, by encapsulation and surface coating, or by covalent functionalization are described. The application of molecular modeling methods to elucidate the selector–selectand interaction is discussed. An application for enantiomeric impurity determination is also considered.

A capillary electrophoresis method for the determination of the linagliptin enantiomeric impurity

Linagliptin is a highly specific, long-acting inhibitor that is used as an orally administrable agent for type-2 diabetes treatment. Because only the R-enantiomer is of clinical use, we developed a capillary electrophoresis method for the determination of the enantiomeric impurity of this compound. Carboxymethyl-β-cyclodextrin was selected as the chiral selector for the separation of linagliptin enantiomers. Design of experiments and desirability functions were used for the analytical optimization, which was focused on understanding and improving the electrophoretic process. The effects of significant parameters (background electrolyte concentration and pH, cyclodextrin concentration, temperature, and voltage) were thoroughly investigated. The complete separation of linagliptin and its enantiomeric impurity with baseline resolution was achieved within 10min on an uncoated fused-silica capillary (50μm inner diameter, 365μm outer diameter, 64.5/56cm in total/ effective length) maintained at 25°C, under an applied voltage of 28.0kV. The background electrolyte contained 70mM sodium acetate and 4.7mM carboxymethyl-β-cyclodextrin, and the pH was adjusted to 6.10. The method was validated, and a limit of quantitation of 0.05% for the impurity was estimated.

Investigation of the synergistic effect of chiral ionic liquids as additives in non-aqueous capillary electrophoresis for enantioseparation

In this work, tetraalkylammonium amino acid ionic liquids (TAA-AAILs) were first applied to non-aqueous capillary electrophoresis (NACE) to establish synergistic systems with a conventional chiral selector, native β-cyclodextrin (β-CD). Excellent enantioseparations of some dansyl-amino acid (Dns-AA) samples were achieved. A series of comparison experiments and a molecular docking study were performed to validate the synergistic effect of TAA-AAILs and β-CD in NACE. Several interesting results were observed compared with previously reported chiral ILs-related aqueous CE studies. In particular, the direct enantioselectivity of TAA-AAILs was observed for the first time by using it as sole chiral selector in NACE. This was an encouraging finding because it was the first direct and convincing evidence that AAILs were able to participate in the enantiorecognition process in the conventional chiral selectors-based synergistic systems. The new TAA-AAILs synergistic NACE system was further optimized in terms of alkyl chain length, TAA-AAILs concentration, β-CD concentration, electrolyte composition and applied voltage, etc. Best enantioseparations of Dns-AAs were obtained when 100 mM β-CD and 10 mM tetramethylammonium-l-arginine (TMA-l-Arg) were added in an NMF buffer containing 50 mM Tris and 35 mM CA (apparent pH 7.85) under UV detection. The applied voltage was set at 30 kV. The method was then successfully employed for the determination of enantiomeric impurities of a real AA sample. This work proved that the use of chiral ILs as additives in NACE is a promising approach for enantioseparation.

Kissing interactions for the design of a multicolour fluorescence anisotropy chiral aptasensor

The development of enantioselective assays and sensors has received much attention for the determination of enantiomeric impurities. Herein, we demonstrated that the previously reported aptamer kissing complex (AKC) assay strategy can be implemented for designing a chiral tool that allows both the simultaneous enantiomer quantification and the enantiopurity analysis. D- and L-arginine vasopressin (AVP) were employed as model enantiomeric targets. The D- and L-AVP engineered aptamers (aptaswitch) were used as recognition units whereas the Fluorescein or Texas Red labelled D- and L-hairpin probes (aptakiss) served as probes of the enantiomer-dependent AKC formation. The orthogonal fluorescence anisotropy signaling scheme at two different emission wavelengths permitted the concomitant sensing of the AVP enantiomers in a single sample, under a high-throughput microplate format. It was also shown that the AKC-based enantioselective sensor allowed the enantiomeric impurity detection at a level as low as 0.01%.

Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride.

Besifloxacin is a unique chiral broad-spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R-form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S-isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high-performance liquid chromatography (HPLC) method for determination of R-besifloxacin and S-besifloxacin (BES impurity A) was developed and validated for in-process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD-H (250 x 4.6mm, 5μm) column using n-heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290nm. The resolution between S-isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal-to-noise ratio of 3 and 10 the LOD of besifloxacin was 0.30μg/mL, while the LOQ was 0.90μg/mL. The calibration curves were linear in the range of 0.9-7.5μg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628-632, 2016. © 2016 Wiley Periodicals, Inc.

Quality by design in the chiral separation strategy for the determination of enantiomeric impurities: Development of a capillary electrophoresis method based on dual cyclodextrin systems for the analysis of levosulpiride

Quality by design (QbD) concepts, in accordance with International Conference on Harmonisation Pharmaceutical Development guideline Q8(R2), represent an innovative strategy for the development of analytical methods. In this paper QbD principles have been comprehensively applied in the set-up of a capillary electrophoresis method aimed to quantify enantiomeric impurities. The test compound was the chiral drug substance levosulpiride (S-SUL) and the developed method was intended to be used for routine analysis of the pharmaceutical product. The target of analytical QbD approach is to establish a design space (DS) of critical process parameters (CPPs) where the critical quality attributes (CQAs) of the method have been assured to fulfil the desired requirements with a selected probability. QbD can improve the understanding of the enantioseparation process, including both the electrophoretic behavior of enantiomers and their separation, therefore enabling its control. The CQAs were represented by enantioresolution and analysis time. The scouting phase made it possible to select a separation system made by sulfated-β-cyclodextrin and a neutral cyclodextrin, operating in reverse polarity mode. The type of neutral cyclodextrin was included among other CPPs, both instrumental and related to background electrolyte composition, which were evaluated in a screening phase by an asymmetric screening matrix. Response surface methodology was carried out by a Doehlert design and allowed the contour plots to be drawn, highlighting significant interactions between some of the CPPs. DS was defined by applying Monte-Carlo simulations, and corresponded to the following intervals: sulfated-β-cyclodextrin concentration, 9–12mM; methyl-β-cyclodextrin concentration, 29–38mM; Britton–Robinson buffer pH, 3.24–3.50; voltage, 12–14kV. Robustness of the method was examined by a Plackett–Burman matrix and the obtained results, together with system repeatability data, led to define a method control strategy. The method was validated and was finally applied to determine the enantiomeric purity of S-SUL in pharmaceutical dosage forms.

Determination of enantiomeric impurity of levamlodipine besylate bulk drug by capillary electrophoresis using carboxymethyl-β-cyclodextrin.

A rapid capillary electrophoresis method using carboxymethyl-β-cyclodextrin (CM-β-CD) as chiral selector was developed and validated for the enantiomeric purity determination of levamlodipine besylate bulk drug. Several parameters for were optimized for a satisfactory enantioresolution, including pH of background electrolyte, the concentration of chiral selector, buffer concentration, capillary temperature and voltage. The highest resolution (Rs = 9.8) was obtained with 4 mM CM-β-CD dissolved in 40 mM phosphate buffer (pH 3.5), at temperature 25 °C and voltage 30 kV, normal polarity. This method was fully validated for the enantiomeric purity determination of the R-amlodipine at the 0.2 % level. The established method was validated in terms of selectivity, LOD and LOQ (0.001 and 0.003 mg mL(-1)), linearity (y = 2.8943x + 0.1386, r (2) = 0.9991), precision and accuracy (95-104 %). Finally, the method was further applied to investigate the enantiomeric purity of levamlodipine in bulk samples.

Determination of enantiomeric impurity in levocetirizine tablets by capillary electrophoresis

To determine enantiomeric impurity in levocetirizine tablets by using capillary electrophoresis. The effects of pH and the concentrations of sulfated-Β-cyclodextrin (S-Β-CD) and buffer salt on chiral resolution were examined with S-Β-CD as chiral selector. A good enantioseparation of cetirizine was achieved with 30 mmol/L NaH2PO4 buffer solution (pH 7.0) containing 20 g/L of S-Β-CD. The method developed in the study is sensitive and reliable for determination of enantiomeric impurity in levocetirizine tablets.

Determination of R(-)- isomer in repaglinide tablets by capillary electrophoresis

To develop a capillary electrophoresis system for enantiomeric impurity test of repaglinide. An uncoated fused silica capillary (50 μm×50 cm, with an effective length of 41 cm) was used. The running buffer was composed of 30 mmol/L NaH2PO4 and 5 mg/ml carboxymethyl-β-cyclodextrin(pH 3.5). Linear range was 2.00-80.00 μg/ml (correlation coefficient was 0.9993). The average recovery rate was 92.5% to 105.0%. The method is simple, accurate and sensitive and it can be used for determination of enantiomeric impurities in repaglinide tablet.

Determination of enantiomeric impurity in besifloxacin hydrochloride by chiral high‐performance liquid chromatography with precolumn derivatization

Besifloxacin hydrochloride is a novel chiral broad-spectrum fluoroquinolone developed for the treatment of bacterial conjunctivitis. R-besifloxacin hydrochloride is used in clinics as a consequence of its higher antibacterial activity. To establish an enantiomeric impurity determination method, some chiral stationary phases (CSPs) were screened. Besifloxacin enantiomers can be separated to a certain extent on Chiral CD-Ph (Shiseido Co., Ltd., Japan), Chiral AGP, and Crownpak CR (+) (Daicel Chemical IND., Ltd., Japan). However, the selectivity and sensitivity were both unsatisfactory on these three CSPs. Therefore, Chiral AGP, Chiral CD-Ph, and Crownpak CR (+) were not used in the enantiomeric impurity determination of besifloxacin hydrochloride. The separation of enantiomers of besifloxacin was further performed using a precolumn derivatization chiral high-performance liquid chromatography method. 2,3,4,6-Tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate was used as the derivatization reagent. Besifloxacin enantiomer derivates were well separated on a C(18) column (250 × 4.6 mm, 5 µm) with a mobile phase that consisted of methanol-KH(2)PO(4) buffer solution (20 mM; pH 3.0) (50:50, v/v). Selectivity, sensitivity, linearity, accuracy, precision, stability, and robustness of this method were all satisfied with the method validation requirement. The method was suitable for the quality control of enantiomeric impurity in besifloxacin hydrochloride.

Chiral separation of metalaxyl and benalaxyl fungicides by electrokinetic chromatography and determination of enantiomeric impurities

The enantiomers of two acylamine fungicides (metalaxyl and benalaxyl) were separated by EKC using CDs as chiral selectors. The use of 15 mM succinyl-γ-CD for metalaxyl and 5 mM succinyl-β-CD for benalaxyl dissolved in a 50 mM 2-morpholinoethanesulfonic acid buffer (pH 6.5), enabled the chiral separation of metalaxyl enantiomers in 11.5 min with a resolution of 3.1 and the enantiomeric separation of benalaxyl in 7.5 min with a resolution close to 15. Under these conditions, the two enantiomers of each of the chiral compound studied were also separated from folpet, very commonly present in fungicide formulations containing metalaxyl or benalaxyl. The analytical characteristics of the two developed methods were studied in terms of precision, linearity, selectivity, limits of detection (LODs) and limits of quantitation (LOQs) showing their suitability for the determination of these compounds in commercial agrochemical formulations. Finally, the development of an in-capillary preconcentration strategy allowed the detection of enantiomeric impurities up to 1.2% in commercial products labeled as enantiomerically pure in metalaxyl-M.

Evaluation of a Vancomycin-Based LC Column in Enantiomeric Separation of Atenolol: Method Development, Repeatability Study and Enantiomeric Impurity Determination

An LC method was developed and validated for the enantioselective separation and enantiomeric impurity quantitation of atenolol. Separation of the atenolol enantiomers on the Chirobiotic V2 (150 mm × 4.6 mm, 5 μm) column was best achieved using a ternary mobile phase of methanol–acetonitrile-triethylamine acetate 0.5% (w/v), pH 4.5 in a ratio of (45:50:5; v/v/v). Good resolution value of R s = 3 was obtained at a flow rate of 1 mL min−1 within a total run time of less than 40 min. Peak identification was achieved using the standard reference of individual enantiomers. The peak of the impurity was eluted in front of the peak of the main enantiomer. Detection was performed by UV at 226 nm. Within and between day’s repeatabilities for both retention time and peak area were investigated at three concentration levels and found to be low. The method was also found to be efficient for the determination of atenolol enantiomeric impurity. An impurity quantitation level of (R)-atenolol down to 0.08% relative to the main enantiomer (S)-atenolol was found possible.

Determination of l- and d-carnitine in dietary food supplements using capillary electrophoresis–tandem mass spectrometry

This paper reports the application of a capillary electrophoresis–electrospray ionisation-tandem mass spectrometry methodology for the unequivocal identification and the quantitative determination of the two enantiomers of the non-protein amino acid carnitine ( l- and d-Carn) in 22 dietary food supplements, including drinks, biscuits, capsules and tablets. MS/MS experiments were optimised to achieve the high sensitivity and selectivity required for food analysis. A comparison of the slopes obtained by the external standard and the standard additions calibration methods indicated the absence of matrix interferences in these food samples. Good precision (RSDs ranged from 2.3% to 4.3% for migration times, and from 2.1% to 10.5% for corrected peak areas) and acceptable accuracy established by means of recovery studies (from 85% to 102%) were obtained. The limit of detection was about 10 ng/mL ( S/ N = 3) enabling the determination of enantiomeric impurities ( d-Carn) up to 0.025% of Carn in foods. This chiral method illustrated is suitable for routine qualitative and quantitative analyses of Carn in foods. The results showed contents for l-Carn comprised from 47% to 115% with respect to the labelled content of l-Carn. Percentages obtained for the d-enantiomer ranged from 0.4% to 5.9%, except for one of the samples analysed, that contained the racemate (49.3% d-Carn). The use of the racemate is not allowed by legislation, which corroborated the high potential of the developed method in the control of the quality and safety of foods containing Carn.

Enantioseparation and impurity determination of the enantiomers of novel phenylethanolamine derivatives by high performance liquid chromatography on amylose stationary phase

Simple and efficient analytical HPLC methods using Chiralpak AS-H as chiral stationary phase were developed for direct enantioseparation of 11 novel phenylethanolamine derivatives. The chromatographic experiments were performed in normal phase mode with n-hexane–ethanol–triethylamine (TEA) as mobile phase. Excellent baseline enantioseparation was obtained for most of compounds. The effects of the concentration of organic modifiers and column temperature were studied for the enantiomeric separation. The mechanism of chiral recognition was discussed based on the relationship between the thermodynamic parameters and structures of compounds. It was found that the enantioseparations were all enthalpy driven, and the tert-butyl groups of compounds had significant influence on the chiral recognition. Trantinterol enantiomers were resolved ( R s = 2.73) within 14 min using n-hexane–ethanol–TEA (98:2:0.1, v/v/v) as mobile phase with a flow rate of 0.8 mL min −1 at 30 °C. The optimized method was validated for linearity, precision, accuracy and stability in solution and proved to be robust. The limits of detection (LOD) and quantification (LOQ) for (+)-trantinterol were 0.15 and 0.46 μg mL −1. The method was applied for enantiomeric impurity determination of (−)-trantinterol bulk samples.

Aptamer-Based Enantioselective Competitive Binding Assay for the Trace Enantiomer Detection

The development of highly enantioselective assays and sensors has received much attention for the determination of enantiomeric impurities at a low level. For chiral compounds, the efficient monitoring of the in selection procedure has allowed the isolation of nucleic acid aptamers which are able to strongly discriminate the target enantiomers. In this paper, we demonstrated for the first time that an aptamer can be successfully used to design a highly enantioselective tool for the trace enantiomer detection. The aptamer-based stereoselective assay was developed using an affinity capillary electrophoresis-based competitive, homogeneous format and an on-capillary mixing approach. Detection of as low as 0.01% of the minor enantiomer in a nonracemic mixture can be achieved, in a short analysis time (<5 min).

Quantification of very low enantiomeric impurity of efaroxan using a dual cyclodextrin system by capillary electrophoresis

A rapid method for the enantiomeric purity determination of efaroxan has been developed by capillary electrophoresis (CE) using a dual cyclodextrin (CD) system. The influence of the nature and the concentration of CDs on separation parameters has been studied. High resolution ( R s = 7) and peak efficiency (104 000–121 000 theoretical plates) values were obtained for efaroxan enantiomers by adding two cyclodextrins, one neutral (7.5 mM DM-β-CD) and the other negatively charged (3 mM CM-β-CD) to the running buffer composed of 100 mM phosphoric acid–triethanolamine (pH 3). These resolution and peak efficiencies values allowed the quantitation of the ( S)-enantiomer of efaroxan at very low enantiomeric excess even if the minor component migrates after the major one. This method was fully validated for the enantiomeric impurity determination of the ( S)-form of efaroxan at the 0.05% level. Calibration curve, expressed by the peak areas ratio versus the enantiomeric purity was linear over the 0.05–1% enantiomeric impurity range ( r 2 = 0.9996). Limits of detection (LOD) and quantification (LOQ), expressed in term of ( S)-enantiomer impurity were 0.02% and 0.05%, respectively. The accuracy of the method at 0.12%, 0.50% and 0.80% enantiomeric impurity levels for the ( S)-form were determined. Recoveries were in 94–102% range for each quality control sample and were determined with good precision (intra-day R.S.D. = 3.54%, inter-day R.S.D. = 5.33%).

Separation of enantiomers of deprenyl with various CDs in CE and the effect of enantiomer migration order on enantiomeric impurity determination of selegiline in active ingredients and tablets

Opposite affinity pattern of enantiomers of the antiparkinsonian chiral drug deprenyl (DEP) was observed towards various neutral and charged derivatives of -CD. The effect of the enantiomer migration order on the LOD of enantiomeric impurity of R-DEP (selegiline) was studied for the standard substances and in the tablets from three different suppliers. The influence of injection mode on the LOD of a minor enantiomeric impurity was also studied and the CE method was compared with the pharmacopoeial HPLC method using a commercially available chiral column Chiralcel OD-H. The optimized CE method was more suitable for low-level enantiomeric impurity determination in selegiline compared to the pharmacopoeial HPLC method.