High-performance enantiomer separation of nonsteroidal anti-inflammatory drugs (NSAIDs) by 3 µm reversed-phase chiral columns and application to the optical purity testing of naproxen drug substances and its formulations.

The enantiomer separation of five nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, ketoprofen (KP) and naproxen (NX), which are included in The Japanese Pharmacopoeia 16th edition (JP16), was investigated by employing four kinds of 3 µm reversed-phase chiral columns (AD-3R, AS-3R, OD-3R and OJ-3R). Except for KP, the enantiomers of four NSAIDs were successfully separated by one of the four columns. Among five NSAIDs, only NX has been used as a single enantiomer (S-form, active form) in the clinical field (JP16); therefore, optical purity testing method of NX is required for its quality evaluation. Among four CSPs, the method was developed by using an AS-3R column, which showed good enantioselectivity for NX enantiomers. By optimizing the conditions, the resolution (Rs) of 2.55 was obtained for NX enantiomers within approximately 6 min. The minor enantiomer R-form eluted before the main active enantiomer S-form. Finally, the developed method was applied to the optical purity testing of NX active pharmaceutical ingredients (APIs), and its formulations (tablet and capsule). Other than the minor enantiomer (R-form, inactive form, 0.21 - 0.78%), normal (not chiral) impurities at levels of 0.01-0.3% were simultaneously separated and determined by the method, showing an excellent separation capability of the method for those impurities including the minor enantiomer. The content uniformity test of the NX tablet according to JP16 was also successfully performed by the method with the AS-3R column. Normal phase separation with two chiral columns (AD-H and OD-H) and capillary electrophoretic (CE) separation were also investigated for five NSAIDs enantiomers to discuss the enantioselectivity.

New trend in the LC separation analysis of pharmaceuticals--high performance separation by ultra high-performance liquid chromatography (UHPLC) with core-shell particle C18 columns--.

This article presents a mini-review of the recent results in the ultra high-performance liquid chromatography (UHPLC) separation of pharmaceuticals by our group. High performance UHPLC separation employing core-shell particle C18 columns was demonstrated. High performance (high theoretical plate number of approximately 20000/10 cm, low theoretical plate height of 5 µm) was obtained without any specific devices in the conventional HPLC apparatus, only through changing detector sampling times and the inner diameter of the connecting tube. High theoretical plate numbers with low column back pressure obtained by the core-shell particle columns enabled fast separation of the analytes. Methanol, which gives high column pressure drops in the reversed-phase mode HPLC compared with acetonitrile, can be used without any trouble. One analysis of the purity testing of diltiazem hydrochloride was performed within 100 s. One analysis in the photostability testing of mecobalamin (vitamin B12 analogue) was successful within 180 s.

Rapid and reliable screening method for detection of 70 pesticides in whole blood by gas chromatography-mass spectrometry using a constructed calibration-locking database.

Pesticide poisoning is one of the most common causes of death by poisoning in Japan, and various kinds of pesticides including organophosphates, carbamates and pyrethroids are listed as causative substances. The purpose of our study was to develop a rapid and reliable screening method for various kinds of pesticides in whole blood by using a unique calibration-locking database and gas chromatography-mass spectrometry. A database of 70 pesticides was constructed using NAGINATA™ software with parameters such as mass spectrum, retention time and qualifier ion/target ion ratio (QT ratio) and calibration curve. Diazepam-d(5) was used as the internal standard for construction of each calibration curve within the range of 0.01-5.0 µg/ml. We examined the applicability of the constructed database by analyzing whole blood samples spiked with 70 pesticides. The pesticides in blood were extracted with hexane under acidic conditions or with an enhanced polymer column (Focus™), subjected to GC-MS, and screened by the pesticides database. Among the 70 pesticides examined, 66 and 62 were successfully identified at the level of 1 and 0.1 µg/ml, respectively, by hexane and 63 and 51 were identified by the Focus column without the use of standard compounds. The time required for data analysis was significantly reduced. Since the established method can produce qualitative and semi-quantitative data without the need for standard substances, this new screening method using NAGINATA™ should be useful for confirming the presence of pesticides in blood in future clinical and forensic cases.

Preparative separation of the saponin lancemaside a from Codonopsis lanceolata by centrifugal partition chromatography.

INTRODUCTION: Lancemaside A is a saponin that inhibits decreases in blood testosterone level and thus prevents or ameliorates symptoms associated with male climacteric disorder. Our initial attempt to preparative isolation of lancemaside A from the saponin fraction of Codonopsis lanceolata roots by a preparative HPLC did not give a clear result. OBJECTIVE: To develop a simple and efficient method for the preparative isolation of lancemaside A from the hot water extract of C. lanceolata roots using centrifugal partition chromatography (CPC). METHODOLOGY: The saponin fraction obtained from the hot water extract of C. lanceolata roots was used as the sample for preparative-scale separation of lancemasides by CPC using n-hexane:n-butanol:methanol:0.1% aqueous formic acid (3:4:1:6, v/v) as the two-phase solvent system. The upper phase (organic phase) of the two-phase solvent system was used as the mobile phase, and 0.5 g of saponin fraction was applied for separation by CPC. Each fraction that was separated by CPC was analysed by HPLC, and the fractions containing each of the separated compounds were pooled together, and then were purified by simple preparative HPLC. RESULTS: The demonstrated separation sequence, hot water extraction, DIAION HP-20 column chromatography, CPC and preparative HPLC, yielded lancemaside A, foetidissimoside A and astersaponin Hb in their pure forms. CONCLUSION: The simple and efficient method for the preparative isolation of lancemaside A along with two other saponins, foetidissimoside A and astersaponin Hb, from the saponin fraction of C. lanceolata was established using CPC.

Neo-clerodane diterpenes from the hallucinogenic sage Salvia divinorum.

Seven new neo-clerodane diterpenes, salvidivins A (2), B, (3), C (4), and D (5), salvinorins H (6) and I (7), and divinatorin [corrected] F (8), along with eight known neo-clerodane diterpenes, salvinorins A (1)-F, divinatorins A and B, and seven other constituents, were isolated from the hallucinogenic sage Salvia divinorum. The structures of 1-7 were elucidated on the basis of 2D NMR spectroscopic studies.