[High performance liquid chromatography/electrospray ionization mass spectrometric characterization of recombinant L-asparaginase II].

AIM: To characterize the primary structure of recombinant L-asparaginase II product. METHODS: The molecular weight of the protein was measured by pneumatically-assisted electrospray ionization mass spectrometry with flow injection mode. Subsequently, tryptic peptide mapping was performed by high performance liquid chromatography on a C8 column with tandem UV and MS detection. An easy-to-use and simple denaturation process with trichloroacetic acid was conducted prior to tryptic digest so as to release the digest resistance from the protein structure. The amino acid sequences of the tryptic peptides were elucidated based on their in-source collision-induced dissociation spectra. RESULTS: The measured molecular mass was different from the theoretical value. Three amino acid variations were unambiguously detected along the peptide backbone derived from the gene-encoding sequence. CONCLUSION: This paper revealed that LC/ESI/MS had provided a promising and robust technique in primary structure analysis and quality control of DNA-derived recombinant protein pharmaceuticals.

[Application of FAM networks in recommendation of mobile phase for RP-HPLC].

AIM: To establish expert system to recommend solvent strength in RP-HPLC by using FAM (fuzzy associate memorizer) networks. METHODS: Symbolic rules reflecting the quantitative relationship among molecular structure, capacity factors and solvent strength in RP-HPLC were extracted from trained radial basis function networks. Then FAM networks consisting of these rules were built to recommend mobile phase strength for RP-HPLC. RESULTS: The validation results of the system by monocomponent and mutilcomponent drug samples were satisfied. CONCLUSION: The mobile phase recommendation system for RP-HPLC analysis of drugs showed reliable performance.

[Application of molecular similarity method in the study of quantitative structure-retention relationship for reversed-phase high performance liquid chromatography of drugs].

Molecular similarity method was applied to the study of quantitative structure-retention relationship (QSRR) for reversed-phase high performance liquid chromatography (RP-HPLC) analysis of drugs. Based on a thorough and systematic study on the molecular structures of 162 drugs, molecular similarity method, which transformed molecular structure parameters to similarity variables, combined with artificial neural network for the study of QSRR for RP-HPLC. The good relationship module reflecting molecular structure, solvent strength and capacity factor was established. Molecular similarity method was successfully used to predict capacity factors (k') and the validation results of 7 drugs were satisfactory. The correlation coefficient of test samples was 0.996, and the residual standard error was 0.244(n = 18). The correlation coefficient of validation samples was 0.992, and the residual standard error was 0.131(n = 7). The application of molecular similarity method in the study of QSRR for RP-HPLC of drugs is satisfactory.

[HPLC/ESI MS and MALDI/TOF MS analysis of microheterogeneity of the N-linked oligosaccharides of recombinant human erythropoietin].

AIM: To elucidate the microheterogeneity of three N-linked oligosaccharide sites of the Chinese-made recombinant human erythropoietin (rHuEPO). METHODS: Glu-C digestion, RP-HPLC separation, online HPLC/electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS: The sialic acid was analyzed directly. Almost every oligosaccharide was acetylated, the acetylation of tetraantennary + 2LacNAc + 4SA and tetraantennary + 2LacNAc + 4SA were reported. CONCLUSION: The acetylation of multi-antennary oligosaccharide will improve the activity of rHuEPO in vivo. The biantennary oligosaccharide was found mainly existing at N-24. For the first time, the carbohydrate structures of each N-linked glycosylated site of Chinese-made rHuEPO were reported.

[Effect of solution pH value on the chelation structure of zinc acexamate by electrospray ionization mass spectrometry].

AIM: To study the effect of solution pH value on the chelation structure of zinc acexamate. METHODS: A series of samples at different solution pH values were prepared by 10% HCl or 1 mol.L-1 NH3.H2O. Then API/TOFMS with electrospray ion source was applied to assay the samples. The nitrogen curtain gas and nebulizer gas were adjusted to a constant flow rate of 0.6 microL.min-1 and 2 microL.min-1 respectively. Samples were infused into the electrospray interface using a 500 microL syringe pump at a flow rate of 5 microL.min-1. Mass spectra were acquired in positive ion modes by scanning over the range of m/z 100-1,000. RESULTS: The chelation structure of zinc acexamate is stable at pH 2.54 and it can be easy to form the ion (M + ZnY)+ (Y = CH3CONH(CH2)5COO-) and (2M + Na)+ in this condition. CONCLUSION: The drug is an effective antiulcer agent. It may decrease the acidity of stomach juice, and form a polymer to protect the ulcer.

Chemical pattern recognition of three Chinese herbal medicines from the genus Stephania lour.

Chemical pattern recognition was applied to three Chinese herbal medicines from the genus Stephania Lour., viz. S. kwangsiensis Lo, S. viridiflavens Lo and M. Yang and S. mashanica Lo and B.N. Chang. Based on the chemical features obtained from HPLC, SIMCA program was carried out and the results showed that the classification accuracy was 100%. In addition, the obtained features showed three major classes by NLM. The results of both methods were consistent with those of plant taxonomical identification. It suggested that chemical pattern recognition could be a helpful method to classify and identify Chinese herbal medicines.

Pharmacokinetic-pharmacodynamic modeling of electroencephalographic effects of midazolam in eight Chinese men.

The effects of midazolam (Mid) on the electroencephalogram (EEG) were related to Mid concentrations in serum in 8 Chinese healthy male volunteers for the assessment of concentration-effect relationship. The total number of waves per second within the frequency range of 12-30 Hz (TNW12-30) in the central-occipital (C1-O1) lead EEG obtained by aperiodic analysis was used as EEG effect of the drug. The PK-PD parameters were calculated by PK-PD software using the sigmoid Emax model. They were: T1(2)keo = 1.3 +/- 0.9 min-1, EC50 = 254 +/- 54 micrograms.L-1, N = 2.9 +/- 0.6. E0 and Emax were calculated from the observed values, being 3.4 +/- 1.3 and 11.4 +/- 2.2 TNW12-30, respectively. Our results showed that the concentration-EEG effect relationship of Mid could be characterized in individual Chinese man using TNW12-30 as a measure of pharmacological response.

Midazolam pharmacokinetics and electroencephalographic changes in eight Chinese men.

Eight Chinese healthy male volunteers aged 27 +/- s 4 a were injected i.v. midazolam (Mid) 15 mg. Blood samples were collected at 0, 2, 5, 7, 10, 20, 30, 45, 60, 90, 120, 180, and 240 min. A HPLC method was established for determining the Mid concentrations in serum. The concentration-time data was fitted with biexponential curve. Pharmacokinetic parameters were: T1/2 alpha = 6.8 +2- 2.5 min, T1/2 beta = 118 +/- 27 min, Vc = 25 +/- 7 L, Cl = 393 +/- 79 ml.min-1, Vdss = 59 +/- 13 L, AUC0-infinity = 39.6 +/- 8.6 g.min.L-1. The electroencephalogram (EEG) showed a decrease in alpha activity and an increase in beta activity. The EEG pattern reverted toward baseline after 2-3 h. Pharmacokinetic and EEG findings suggest that Mid is a preferable anesthesia inducing agent.

[Quantitative analysis of compound injection of aminopyrine by dual-wavelength linear regression spectrophotometry].

A dual-wavelength linear regression spectrophotometry has been introduced and evaluated. Depending on a group of standard mixture solutions the optimal wavelengths and calibration curve can be determined simultaneously by linear regression method. The deviation of absorption resulting from molecular interaction can be calibrated by this method and the results are more accurate. The validity of this method has been confirmed through its use in the analysis of compound injection of antipyrine with satisfactory recoveries. Results obtained by Kalman filtering (KF), partial least squares (PLS) and target factor analysis (TFA) are also given.

[Determination of compound preparation containing unknown absorptive background by UV spectrophotometry].

A novel algorithm of target factor analysis has been developed for detection and correction of unknown absorptive background in multicomponent analysis. The algorithm is based on the property that the estimated spectra can gradually approach the true ones by iterative refinements. Paracetamol and antipyrine contained in compound injection of paracetamol were determined by this method without any preliminary chemical separation. The average recoveries were both 100.0% and the coefficients of variation were 1.1% and 1.0% respectively. The results clearly indicate that the proposed method may also provide a new approach to the analysis of traditional Chinese medicine containing some unknown absorptive components.

Application of pyrolysis-high-resolution gas chromatography-pattern recognition to the identification of the Chinese traditional medicine mai dong.

Pyrolysis-high-resolution gas chromatography-pattern recognition (Py-HRGC-PaRe) was used to develop a potential technique for identifying the Chinese traditional medicine Mai Dong. About 1 mg of crude drug powder was pyrolysed in a furnace pyrolyser and the products were directly carried into a gas chromatograph with an FSOT capillary column (30 m x 0.265 mm I.D.) coated with DB-1701 (df 0.25 micron). The Py-HRGC data were analysed by non-linear mapping PaRe. The results showed that Mai Dong samples could be classified into two categories: Ophiopogon japonicus (L.f.) Ker-Gawl (included in the Chinese Pharmacopoeia) and Liriope spicata.

[Classification of Chinese traditional medicine, moutan (Paeonia suffruticosa Andr.) cortex by means of pyrolysis-high resolution gas chromatography/pattern recognition].

Thirty-eight samples of Moutan (Paeonia suffruticosa Andr.) Cortex obtained from three different regions (Southwest, East and Middle China) and two samples of unknown region were subjected to analysis with pyrolysis-high resolution gas chromatography (Py-HRGC). Each sample was thus characterized by the peak area of 41 peaks in each Py-HRGC profile. Discriminant analysis (DA), PRIMA and SIMCA pattern recognition were used to recognize the 40 x 41 data matrix. These data analysis gave satisfactory results (DA, 100% correct; PRIMA, 100% correct, 92.2% unique; SIMCA, 92.2% correct, 79% unique). The correct classification of Moutan Cortex for the unknown territory was obtained by three pattern recognition methods. The results showed that Py-HRGC/pattern recognition technique might be a potential tool for the identification of Chinese traditional medicine.

[Simultaneous determination of 6 components contained in compound reserpine tablets by Kalman filtering spectrophotometry].

Kalman filtering spectrophotometry was investigated to assay the contents of vitamin B1, vitamin B6, chlordiazepoxide, dihydralazine sulfate, promethazine hydrochloride and hydrochlorothiazide in compound reserpine tablets consisting of 10 components. Absorption proportionality constant for each component was obtained by the application of non-negative least square method. The average recoveries for each were 97-103% with CV% less than or equal to 6.9 except vitamin B1 (n = 11).

[P-matrix spectrophotometric determination of 6 components contained in compound reserpine tablets].

P-matrix method has been developed for the simultaneous determination of vitamin B1, vitamin B6, chlordiazepoxide, dihydralazins sulfate, promethazine hydrochloride and hydrochlorothiazide contained in compound reserpine tablets consisting of 10 components. The optimal wave-length for measurement was successfully selected based on the principle of condition number. The influence of experimental error, number of wavelength and condition number deviation of component concentration from formula were discussed. The average recoveries varied from 97-103% with CV less than or equal to 9.5% (n = 11).

[Simultaneous determination of four components in vitamin B compound tablets by Kalman filtering spectrophotometry].

The four principal components, vitamin B1, B2, B6 and nicotinamide in vitamin B compound tablet show certain instabilities individually and the amounts of these components differ considerably in the tablets, so their simultaneous determination without prior separation is usually difficult. This paper deals with the feasibility of using Kalman filtering spectrophotometry to do so, and makes some efforts concerning the deviation from the Beer-Lambert law due to mutual effects among the acting molecules. The results obtained were comparatively satisfactory both in precision and in accuracy. The average recovery of vitamin B1, B2, B6, and nicotinamide were 100.2 +/- 0.90% (CV), 100.3 +/- 1.7% (CV), 101.1 +/- 3.3% (CV), 99.90 +/- 0.65% (CV) respectively and were better than those in previous reports. All these show that the use of Kalman filtering spectrophotometry to the assay of vitamin B compound tablets is feasible.