Real-world corticosteroid use in severe pneumonia: a propensity-score-matched study.

BACKGROUND: Community-acquired pneumonia (CAP) is a leading cause of morbidity and mortality worldwide despite correct antibiotic use. Corticosteroids have long been evaluated as a treatment option, but heterogeneous effects on survival have precluded their widespread implementation. We aimed to evaluate whether corticosteroids might improve clinical outcomes in patients with severe CAP and high inflammatory responses. STUDY DESIGN AND METHODS: We analyzed two prospective observational cohorts of patients with CAP in Barcelona and Rome who were admitted to intensive care with a high inflammatory response. Propensity score (PS) matching was used to obtain balance among the baseline variables in both groups, and we excluded patients with viral pneumonia or who received hydrocortisone. RESULTS: Of the 610 patients admitted with severe CAP, 198 (32%) received corticosteroids and 387 had major criteria for severe CAP. All patients had a baseline serum C-reactive protein above 15 mg/dL. Patients who received corticosteroids were more commonly male, had more comorbidities (e.g., cancer or chronic obstructive pulmonary disease), and presented with significantly higher sequential organ failure assessment scores. Eighty-nine patients met major severity criteria (invasive mechanical ventilation and/or septic shock) and were matched per group. Twenty-eight-day mortality was lower among patients receiving corticosteroids (16 patients, 18%) than among those not receiving them (28 patients, 31%; p = 0.037). After PS matching, corticosteroid therapy reduced the 28-day mortality risk in patients who met major severity criteria (hazard ratio (HR) 0.53, 95% confidence interval (CI) 0.29-0.98) (p = 0.043). In patients who did not meet major severity criteria, no benefits were observed with corticosteroid use (HR 0.88 (95%CI 0.32-2.36). CONCLUSIONS: Corticosteroid treatment may be of benefit for patients with CAP who have septic shock and/or a high inflammatory response and requirement for invasive mechanical ventilation. Corticosteroids appear to have no impact on mortality when these features are not present.

Mesenchymal stem/stromal cell-based therapies for severe viral pneumonia: therapeutic potential and challenges.

Severe viral pneumonia is a significant cause of morbidity and mortality globally, whether due to outbreaks of endemic viruses, periodic viral epidemics, or the rarer but devastating global viral pandemics. While limited anti-viral therapies exist, there is a paucity of direct therapies to directly attenuate viral pneumonia-induced lung injury, and management therefore remains largely supportive. Mesenchymal stromal/stem cells (MSCs) are receiving considerable attention as a cytotherapeutic for viral pneumonia. Several properties of MSCs position them as a promising therapeutic strategy for viral pneumonia-induced lung injury as demonstrated in pre-clinical studies in relevant models. More recently, early phase clinical studies have demonstrated a reassuring safety profile of these cells. These investigations have taken on an added importance and urgency during the COVID-19 pandemic, with multiple trials in progress across the globe. In parallel with clinical translation, strategies are being investigated to enhance the therapeutic potential of these cells in vivo, with different MSC tissue sources, specific cellular products including cell-free options, and strategies to 'licence' or 'pre-activate' these cells, all being explored. This review will assess the therapeutic potential of MSC-based therapies for severe viral pneumonia. It will describe the aetiology and epidemiology of severe viral pneumonia, describe current therapeutic approaches, and examine the data suggesting therapeutic potential of MSCs for severe viral pneumonia in pre-clinical and clinical studies. The challenges and opportunities for MSC-based therapies will then be considered.

Comparison of liquid chromatography and capillary electrophoresis methods for quantification of sodium residuals.

Liquid chromatography (LC) and capillary electrophoresis (CE) methods were developed to perform the determination of residual sodium in mother liquors and successive washes of an active pharmaceutical ingredient (API). The addition of sodium chloride to the product solution results in rapid and complete crystallization of the API. The LC method was coupled to evaporative light scattering detection (ELSD) while the CE approach was based on indirect UV detection. Both methods were fully validated. Selectivity, response function, trueness, precision, accuracy, linearity and limits of detection (LOD) and quantification (LOQ) were the criteria investigated. The LC-ELSD method was found to be more sensitive than the CE/indirect UV approach. The methods were found to be valid over concentration ranges of 62-500 and 235-1500 ppm for the LC and the CE methods, respectively. Both methods were compared and used for the determination of actual samples coming from different batches of the same API chemical synthesis.

A new validation approach applied to the GC determination of impurities in organic solvents.

Organic solvents such as methanol, acetone, dichloromethane or toluene are frequently used in the pharmaceutical industry. The manufacturing of new active pharmaceutical ingredients (APIs) under GMP conditions commands to control adequately the quality of the different ingredients happening in the synthesis. Organic solvents have therefore to be controlled and their purity has to be determined before any GMP synthesis. A selective gas chromatography (GC) method has been developed to determine the purity of acetone, dichloromethane, methanol and toluene. Using this method, the main contaminants of each organic solvent can be quantified. Moreover, the developed method allows the simultaneous determination of ethanol, isopropanol, chloroform, benzene, acetone, dichloromethane, methanol and toluene. Propionitrile was used as the internal standard. The separation was obtained on a CP-SIL 8-CB low bleed/MS column (60 m x 0.32 mm i.d.x1.0 microm coating thickness). The GC method was fully validated using a new approach based on the accuracy profile as a decision tool. The determination of beta-expectation tolerance intervals for the estimation of total error - including both bias and precision - is used to better reflect the actual performances of the method, which is definitively the objective of the validation. The different validation criteria such as selectivity, response function, trueness, precision, accuracy, linearity or limits of detection and quantification were considered. The method was found to be able to quantitate with a good accuracy impurities around the 0.1% (v/v) concentration level for the different solvents.

The transfer of a LC-UV method for the determination of fenofibrate and fenofibric acid in Lidoses: use of total error as decision criterion.

Two new statistical approaches to assess the validity of the transfer of a LC-UV method for the determination of fenofibrate and fenofibric acid were investigated and compared to the conventional approaches generally used in this domain. These new approaches, namely the Tolerance Interval and the Risk approaches, are based on the simultaneous evaluation of the systematic (or trueness) and random (or precision) errors of the transfer into a single criterion called total error (or accuracy). The results of the transfer showed that only the total error based approaches fulfilled the objective of an analytical method transfer, i.e. to give guarantees that each future measurement made by the receiving laboratory will be close enough to the true value of the analyte in the sample. Furthermore the Risk approach was the most powerful one and allowed the estimation of the risk to have future measurements out of specification in the receiving laboratory, therefore being a risk management tool.

Validation of a liquid chromatographic-tandem mass spectrometric method for the determination of loperamide in human plasma.

A sensitive and selective method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the quantitative determination of loperamide in human plasma. Automated solid-phase extraction (SPE) on disposable extraction cartridges (DEC) is used to isolate the compounds from the biological matrix and to prepare a cleaner sample before injection and analysis in the LC-MS/MS system. After conditioning, the plasma sample is loaded on the DEC filled with endcapped ethyl silica (C2(EC)) and washed twice with water. The analytes are therefore eluted by dispensing methanol. The eluate is then collected and added with ammonium acetate solution in order to inject an aliquot of this final extract in the LC-MS/MS system. On-line LC-MS/MS system using atmospheric pressure chemical ionization (APCI) has been developed for the determination of loperamide. The separation is obtained on a octadecylsilica based stationary phase using a mobile phase consisting in a mixture of methanol and 5mM ammonium acetate solution (25:75, v/v). Clonazepam is used as internal standard (IS). The MS/MS ion transitions monitored are m/z 477--> 266 and 316--> 270 for loperamide and clonazepam, respectively. The most appropriate regression model of the response function as well as the limit of quantitation were first selected during the pre-validation step. These latter criteria were then assessed during the formal validation step. The limit of quantitation (LOQ) was around 50 pg/ml for loperamide. The method was also validated with respect to recovery, precision, trueness, accuracy and linearity.

Determination of the enantiomers of 3-tert.-butylamino-1,2-propanediol by high-performance liquid chromatography using mass spectrometric detection.

The chiral synthesis of beta-blockers such as (S)-timolol requires a sensitive analytical method for the enantioseparation of its intermediate, 3-tert.-butylamino-1,2-propanediol, in the ng/ml range. The method developed is based on on-line normal-phase LC-MS-MS using a chiral stationary phase and an atmospheric pressure chemical ionization (APCI) interface. The MS detection of 3-tert.-butylamino-1,2-propanediol was first optimized with a pneumatically-assisted electrospray interface (ionspray). The APCI interface was then selected for LC-MS-MS because of the incompatibility of electrospray with n-hexane. The method was validated for both enantiomers in the 25-500 ng/ml concentration range.

Automated determination of verapamil and norverapamil in human plasma with on-line coupling of dialysis to high-performance liquid chromatography and fluorometric detection.

A fully automated method for the simultaneous determination of verapamil and its main metabolite norverapamil in human plasma is described. This method is based on on-line sample preparation using dialysis followed by clean-up and enrichment of the dialysate on a precolumn and subsequent HPLC analysis with fluorometric detection. All sample handling operations were performed automatically by a sample processor equipped with a robotic arm (ASTED system). The plasma samples were dialysed on a cellulose acetate membrane (cut-off: 15 kD) and the dialysate was purified and enriched on a short pre-column filled with cyanopropyl silica. Before starting dialysis, this trace enrichment column (TEC) was first conditioned with the HPLC mobile phase and then with pH 3.0 acetate buffer. 370 microliters of plasma sample spiked with the internal standard (gallopamil) were dialysed in the static-pulsed mode. The solution at the donor side was pH 3.0 acetate buffer containing Triton X-100 while the acceptor solution was made of the same acetate buffer. When dialysis was discontinued, the analytes were desorbed from the TEC by the HPLC mobile phase and transferred to the C18 analytical column by means of a switching valve. This mobile phase consisted of a mixture of acetonitrile, pH 3.0 acetate buffer and 2-aminoheptane. The influence of different parameters of the dialysis process on the recovery of verapamil and norverapamil has been studied. The effect of the volume, the aspirating and dispensing flow-rates of the dialysis solution has been investigated. The recoveries of verapamil and norverapamil in plasma were close to 75% and the limits of quantification were 5 ng/ml for both analytes. The method was found to be linear in the concentration range from 5 to 500 ng/ml (r2: 0.9996 for both analytes). The intra-day and inter-day reproducibilities at a concentration of 100 ng/ml were 2.3% and 5.6% for verapamil and 1.7% and 5.1% for norverapamil, respectively.

Simultaneous determination of methylphenobarbital enantiomers and phenobarbital in human plasma by on-line coupling of an achiral precolumn to a chiral liquid chromatographic column.

A fully automated liquid chromatographic (LC) method for the simultaneous determination of methylphenobarbital enantiomers and phenobarbital in human plasma has been developed. The method is based on the use of a precolumn packed with an internal-surface reversed-phase packing material (LiChrospher ADS) for sample clean-up coupled to LC analysis on a cellulose tris(4-methylbenzoate) based chiral stationary phase (Chiralcel OJ-R). A 100-microliter plasma sample was injected directly on the precolumn packed with LiChrospher RP-18 ADS using a mixture of pH 5.0 phosphate buffer-methanol (97:3, v/v) as washing liquid. The analytes were then eluted in the back-flush mode with the LC mobile phase. The enantiomeric separation of methylphenobarbital was achieved on Chiralcel OJ-R). The retention times were modelled using a D-optimal design with ten experimental points in order to optimise the LC mobile phase for the separation of phenobarbital from the enantiomers of mephobarbital. The factors selected were the acetonitrile content, the pH and the sodium perchlorate concentration in the mobile phase. A Derringer's desirability function was used to find an optimal and robust solution within the experimental domain. The mobile phase selected consisted of a mixture of pH 7.0 phosphate buffer-acetonitrile (60:40, v/v). The elution profiles of phenobarbital, methylphenobarbital and blank plasma samples on the precolumn and the time needed for analyte transfer from the precolumn to the analytical column were then determined. Finally, the method developed was validated.

Quantitative analysis of N-acetylcysteine and its pharmacopeial impurities in a pharmaceutical formulation by liquid chromatography-UV detection-mass spectrometry.

A new method for the simultaneous determination of N-acetylcysteine and its pharmacopeial impurities, cysteine, cystine, N,N'-diacetylcystine and N,S-diacetylcysteine in an effervescent tablet has been developed. The method is based on on-line LC-UV-MS using a pneumatically-assisted electrospray interface (ionspray). The stability of the thiol moieties of the analytes was ensured by the acidic pH of the LC mobile phase. Quantitation of N-acetylcysteine was performed with UV detection to avoid ion-source overloading effect due to its higher concentration, whereas the impurities could be easily separated and quantified in MS. The method was validated in terms of stability, linearity, precision and accuracy.

Determination of molsidomine and its active metabolite in human plasma using liquid chromatography with tandem mass spectrometric detection.

Pharmacokinetic studies of molsidomine require a sensitive analytical method to allow the determination of concentrations of this compound and its active metabolite 3-morpholinosydnonimine (Sin-1) in the ng/ml range in plasma. The method developed is based on on-line LC-MS-MS using pneumatically assisted electrospray ionisation as an interface, preceded by off-line solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (automated sample preparation with extraction cartridges; ASPEC system). The DEC, filled with phenyl-modified silica, was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol containing formic acid (0.2%) and water. The liquid chromatographic separation of molsidomine and Sin-1 was achieved on an RP-8 stationary phase (5 microns). The mobile phase was a mixture of methanol-water-formic acid (65:35:0.1, v/v/v). The HPLC system was then coupled to a MS-MS system with an atmospheric pressure ionisation interface in the positive ion mode. The chromatographed analytes were detected in the multiple reaction monitoring mode. The MS-MS ion transitions monitored were (m/z) 243-->86 for molsidomine and 171-->86 for Sin-1. The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 74 +/- 3 and 55 +/- 5% for molsidomine and Sin-1, respectively. The method was found to be linear in the 0.5-50 ng/ml concentration range for the two analytes (r2 = 0.999 for both molsidomine and Sin-1). The mean RSD values for repeatability and intermediate precision were 3.4 and 4.8% for moldsidomine and 3.1-7.7% for the metabolite. The method developed was successfully used to investigate the bioequivalence of oral doses of molsidomine between a generic tablet and a reference product.

Fully automated method for the liquid chromatographic-tandem mass spectrometric determination of cyproterone acetate in human plasma using restricted access material for on-line sample clean-up.

A new automated method for the quantitative analysis of cyproterone acetate (CPA) in human plasma has been developed using on-line solid phase extraction (SPE) prior to the LC-MS/MS determination. The method was based on the use of a pre-column packed with internal-surface reversed-phase material (LiChrospher RP-4 ADS, 25 mm x 2 mm) for sample clean-up coupled to LC separation on an octadecyl silica stationary phase by means of a column switching system. A 30 microl plasma sample volume was injected directly onto the pre-column using a mixture of water, acetonitrile and formic acid (90:10:0.1 (v/v/v)) adjusted to pH 4.0 with diluted ammonia as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase consisting of water, methanol and formic acid (10:90:0.1 (v/v/v)). The dispensing flow rates of the washing liquid and the LC mobile phase were 300 microl min(-1). Medroxyprogesterone acetate (MPA) was used as internal standard. The MS ionization of the analytes was achieved using electrospray (ESI) in the positive ion mode. The pseudomolecular ionic species of CPA and MPA (417.4 and 387.5) were selected to generate daughter ions at 357.4 and 327.5, respectively. Finally, the developed method was validated according to a new approach using accuracy profiles as a decision tool. Very good results with respect to accuracy, detectability, repeatability, intermediate precision and selectivity were obtained. The LOQ of cyproterone acetate was 300 pg ml(-1).

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry.

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.

Development and validation of a liquid chromatographic method for the determination of amlodipine residues on manufacturing equipment surfaces.

In the pharmaceutical industry, an important step consists in the removal of possible drug residues from the involved equipment and areas. The cleaning procedures must be validated and the methods to determine trace amounts of drugs have therefore to be considered with special attention. A high performance liquid chromatographic method for the determination of amlodipine residues in swab samples was developed and validated in order to control a cleaning procedure. The swabbing procedure was optimized in order to obtain a suitable recovery of amlodipine from stainless steel. A mean recovery close to 90% was obtained when two swabs moistened with methanol were used. The residual amlodipine was chromatographed at 25 degrees C in the isocratic mode on a RP-18 stationary phase using a mobile phase consisting of acetonitrile, methanol and pH 3.0 triethylamine solution (15:35:50 v/v/v). UV detection was performed at 237 nm. The method was shown to be selective and linear into the concentration range varying from 0.39 to 1.56 microg/ml. Accuracy and precision of the method were also studied. The limits of detection and quantitation were evaluated to be 0.02 and 0.08 microg/ml, respectively. The stability of amlodipine at different steps of the sampling procedure and the precision of the swabbing procedure were also investigated.

Sensitive determination of buprenorphine and its N-dealkylated metabolite norbuprenorphine in human plasma by liquid chromatography coupled to tandem mass spectrometry.

A highly sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the quantitative determination of buprenorphine and its active metabolite norbuprenorphine in human plasma. Automated solid phase extraction (SPE) on disposable extraction cartridges (DEC) is used to isolate the compounds from the biological matrix and to prepare a cleaner sample before injection and analysis in the LC-MS/MS system. After conditioning, the plasma sample (1.0 ml) is loaded on the DEC filled with octyl silica (C8) and washed with water. The analytes are, therefore, eluted by dispensing methanol containing 0.1% of acetic acid. The eluate is collected and evaporated to dryness. The residue is dissolved in mobile phase and an aliquot is injected in the LC-MS/MS system. On-line LC-MS/MS system using atmospheric pressure chemical ionization (APCI) has been developed for the determination of buprenorphine and norbuprenorphine. The separation is obtained on a RP-18 stationary phase using a mobile phase consisting in a mixture of methanol and 50 mM ammonium acetate solution (50:50, v/v). Clonazepam is used as internal standard (IS). The MS/MS ion transitions monitored are m/z 468-->468, 414-->414 and 316-->270 for buprenorphine, norbuprenorphine and clonazepam, respectively. The method was validated regarding recovery, linearity, precision and accuracy. The limits of quantification (LOQs) were around 10 pg/ml for buprenorphine and 50 pg/ml for norbuprenorphine.

Determination of meprobamate in pharmaceutical dosage forms also containing carbromal by liquid chromatography and indirect photometric detection.

In a pharmaceutical form also containing carbromal, meprobamate could not be quantified selectively by classical methods described in pharmacopoeias due to a significant interference from carbromal. Consequently, reversed-phase HPLC methods have been developed to separate the two active ingredients using indirect photometric detection to visualize and determine meprobamate which has very poor chromophoric properties. Different parameters influencing the sensitivity of the indirect response, such as the nature of the highly absorbing compound added to the mobile phase (the marker) as well as the methanol content and the pH of this phase, have been studied. Two chromatographic systems containing benzoic acid or cinnamic acid as the marker, have been optimized and validated. Good linearity and reproducibility have been obtained with both systems but the cinnamic acid method has the advantage that meprobamate and carbromal can be determined simultaneously at 273 nm.

Enantioselective determination of oxprenolol in human plasma using dialysis coupled on-line to reversed-phase chiral liquid chromatography.

A fully automated method for the determination of the enantiomers of oxprenolol in human plasma was developed, involving dialysis through a cellulose acetate membrane, clean-up and enrichment of the dialysate on a short precolumn and subsequent chiral liquid chromatographic (LC) analysis. All sample handling operations were executed automatically by a sample processor equipped with a robotic arm (ASTED system). The trace enrichment column (TEC) was packed with octadecylsilica. After conditioning of the TEC with the LC mobile phase and pH 3.0 acetate buffer. After the enrichment step, the analyte was transferred by the LC mobile phase to the analytical column by means of a switching valve. The influence of different parameters of the dialysis process on the recovery of oxprenolol was first investigated using achiral LC conditions. The volume as well as the aspirating and dispensing flow rates of the acceptor solution were the main parameters studied. Oxprenolol was separated on a C18 stationary phase used for the enantioseparation of oxprenolol was a Chiralcel OD-R column which contained cellulose tris (3,5-dimethylphenylcarbamate) coated on silica as chiral selector. The corresponding mobile phase consisted of a mixture of pH 6.0 phosphate buffer containing NaClO4 at 0.45 M concentration and acetonitrile (70:30 v/v). UV detection was performed at 273 nm. The method developed was validated. Recoveries for each enantiomer of oxprenolol were about 80%. The method was found to be linear in the 50-2500 ng ml-1 concentration range (r2 = 0.999 for both enantiomers) and good results with respect to intra- and inter-day reproducibility as well as accuracy were obtained.

Determination of verapamil and norverapamil in human plasma by liquid chromatography: comparison between a liquid-liquid extraction procedure and an automated liquid-solid extraction method for sample preparation.

A conventional liquid-liquid extraction (LLE) procedure with high-performance liquid chromatography (HPLC) has been developed for the determination of verapamil and its main metabolite, norverapamil, in plasma. After addition of the internal standard, plasma samples were basified with phosphate buffer (pH 9.0) and extracted with a mixture of cyclohexane-dichloromethane. After centrifugation, the organic layer was separated and the analytes were extracted back into a 0.1 N sulphuric acid solution containing 2-aminoheptane. An aliquot of this aqueous phase was then injected directly onto the HPLC column. This LLE procedure has been compared with an automated liquid-solid extraction (LSE) method that has been developed in parallel. Good linearity was obtained using both extraction methods. The absolute recoveries for the two analytes were ca 95% with the automated LSE procedure and slightly lower (ca 84%) for the LLE method. The automated method gives better results with respect to detectability and precision, but the LLE procedure is simpler to develop, requires much less expensive equipment, and remains a useful alternative when the number of samples to be analysed is limited.

Simultaneous determination of pirlindole enantiomers and dehydropirlindole by chiral liquid chromatography.

Liquid chromatography was employed for the determination of pirlindole enantiomers and its oxidation product dehydropirlindole (DHP). The direct separation of pirlindole enantiomers and DHP was achieved on a cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralcel OD-R). Acetonitrile was used as the organic modifier and sodium perchlorate was used as an ionic additive in the mobile phase. The influence of acetonitrile and sodium perchlorate concentrations on enantioselectivity and achiral selectivity towards DHP was investigated in order to find suitable conditions for the determination of low amounts of each analyte. The mobile phase selected consisted of a mixture of acetonitrile and phosphate buffer (pH 5.0) containing sodium perchlorate (0.05 M) (35:65, v/v) and the UV detector was set at 220 nm. The method developed was validated and was found to be linear in the 0.1-5 microg ml(-1) range (r2 = 0.999 for the three compounds). Repeatability and the intermediate precision for the three analytes at a concentration of 0.1 microg ml(-1) were about 3 and 4%, respectively. This concentration corresponds to the quantification of 0.1% for the minor enantiomer. Actual determinations of enantiomeric purity for single enantiomers of pirlindole were performed.

Enantiomeric separation of pirlindole by liquid chromatography using different types of chiral stationary phases.

The enantioseparation of pirlindole by liquid chromatography (LC) was investigated using three different chiral stationary phases (CSPs) containing either cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD-R), ovomucoid (OVM) or beta-cyclodextrin (beta-CD). The effects of the mobile phase pH on retention, enantioselectivity and resolution were studied. Methanol and acetonitrile were tested as organic modifiers while the influence of the addition to the mobile phase of sodium alkanesulfonates or sodium perchlorate was also investigated. Sodium perchlorate was only used on the Chiralcel OD-R column while sodium alkanesulfonates were tested as mobile phase additives on the three kinds of CSPs. The enantioseparation of pirlindole could be obtained on all CSPs tested, the best results with respect to chiral resolution being achieved on the Chiralcel OD-R and the OVM columns. The use of sodium octanesulfonate (NaOS) was found to improve the enantioseparation of pirlindole on the OVM column while enantioselectivity was considerably enhanced by addition of sodium perchlorate on the Chiralcel OD-R column.