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Interlaboratory study of the benzylpenicillin by liquid chromatography
Relating
to Invited Lecture 2-Analytical
System .wimbi& teata (SSTs) are wentid for enwring reliability and robustneaaof HPLC systems+and i&l con6dencc in results obtained Since theHPU:cohumristhcmortimporUntprrtofa~yrtm5bothchiraland sundrrd~~thwldkmonitoredonarcgularmd~umtbasis Inthiswrym~levdofpafonarncelrndiat~ofthesystem(and HPLC cohlmtt) can be enaured.
Chemistry
Sl69
(P3.001-P3.030)
A modified hepatitisB surface antigen carryingthe pre-S2 region (TGP-943) was prnducedin yeast using recombinantDNA technology and developed as a new type of hepatitisB vaccine. In order to conduct peptide mapping to confirm the amino acid sequence, conditions for enzymatic digestion and cyanogenbromide
ChinlHPLCp~bynmtrrofthdrpvityandmethodof~~~eare genmlly expaakve aad often f?ag& for example protein HPLC columns are highly awceptibie to suddat and wcxpected deterioration. For these reasons, chiral cohUnn!+in particular require carefill tnonitoring.
cleavage were optimized, and an RP-HPLC method that can directly
A number of SSTafor chirsl HPLC systana utilising a selc&on of common attd aovd elkI HPLC columns are preaawd. These columnsinclude various cycMutrin tmd protein phased.Common drug analytes have been chosen for these SSTs and include: propranolol; veraparnil;dprenolol; tioconazole, and benzoii.
trypsin and/or lysylendopeptidaseor cleaved with cyanogenbromide
analyze hydrophobic peptides dissolved in a sodium dodecylsulfate solutionwas developed. Reduced and S-carboxymethylatedTGP-943 was digested with
The resulting peptides were separated and fractionatedby RP-HPLC and then characterized by amino acid analysis. The resultssupported the amino acid compositionof each peptide expected from the cDNA sequence. Furthermore, it was confirmed that TGP-943 carries sugar
This work haa aigni6cant implicationsfor assuranceof robustnessin regulatory aubmisaiona
chains using digestionwith glycosidase,and the amino acid composition of the glycopeptidesrevealed that the sugar chains are mainly in the pre-S2 region.
FT-Raman spectroscopy has proved to be a useful technique for the study of the molecular basis of normal, healthy and abnormal, diseased human tissue’. Verrucae or warts are contagious, benign tumours resulting from a viral infection which affects only the skin, appearing as small papules or nodules on the skin or mucous membrane. Here we have used FT-Raman spectroscopy to study excised verrucae which had been successfully treated with a local application of a salicylic acid ointment. A liquid chromatography method for analysis of benzytpenicillin was examined in a daborative study. The method comprises an isocratii part, which is used in the assay. When the isocratic part is combined with gradient elution. the method is suiteble for purity control. The method prescribes the use of Cl8 stationary phase with 5 pm particle size and column dimensions of 25 x 0.46 cm i.d. a flow rate of 1.0 ml min-‘, an injector with a loop of about 20 pl and a UV detector set at 225 nm. Two mobile phases were used. Mobile phase A was a mixture of 0.5 M phosphate buffer pH 3.5~methanol-water (10:30:60. v/vhr) and mobile phase B was a mixture of the same components with a ratio (10:50:40. v/v/v). For the assay, isocratic elution was used with a mobile phase ratio A:B of 70:30. For the related substances test, isocratic elutiin combined with gradient elution was petformsd as follows: after isocratic elution of the benzylpenicillin peak with a mobile phase ratio A:8 used in the assay, a linear gradient elution was started to reach a mobile phase ratio A:B of 0:lOO over a period of 20 min. this ratio was held for 15 min. then the column was equilibrated with a mobile phase ratio A:B of 70:30 during 15 min. Five samples of benzylpenicillin (sodium and potassium salt) with varying purity were analysed. The main component and the impurities were determined. The results will be shown together with an anaiysii of variance and calculated estimates for the repeatability and reproducibilii of the method.
FT-Raman spectra were recorded using a Bruker FRA 106 Raman module mounted on a Bruker IFS 66 optical system, using NIR (1.064 urn) laser excitation. Spectra were typically recorded over 1000 scans at 4 cm“ at a laser power of 400-500 mW. Differences in the molecular structure of the stratum comeum across the surface of the sample were observed, and by comparison with normal and hyperkeratotic (callus) stratum wmeum it was concluded that the tissue around the edge of the sample was typically healthy skin. In the centre of the verruca, the molecular structure of the skin is altered and evidence for the presence of salicylic acid is observed. The salicylic acid is not observed in a pure crystalline state but appeared to be present wmplexed within the skin. No evidence for the presence of the other ointment components, lactic acid and flexible wllodion was found in the spectrum of the centre of the verruca This approach may help elucidate the molecular therapeutic agents acting on skin disorders.
basis for
‘Edwards, H.G.M. et al., J.Mol.Structure.. 347, 379 (1995)
to Invited Lecture 2-Analytical
System .wimbi& teata (SSTs) are wentid for enwring reliability and robustneaaof HPLC systems+and i&l con6dencc in results obtained Since theHPU:cohumristhcmortimporUntprrtofa~yrtm5bothchiraland sundrrd~~thwldkmonitoredonarcgularmd~umtbasis Inthiswrym~levdofpafonarncelrndiat~ofthesystem(and HPLC cohlmtt) can be enaured.
Chemistry
Sl69
(P3.001-P3.030)
A modified hepatitisB surface antigen carryingthe pre-S2 region (TGP-943) was prnducedin yeast using recombinantDNA technology and developed as a new type of hepatitisB vaccine. In order to conduct peptide mapping to confirm the amino acid sequence, conditions for enzymatic digestion and cyanogenbromide
ChinlHPLCp~bynmtrrofthdrpvityandmethodof~~~eare genmlly expaakve aad often f?ag& for example protein HPLC columns are highly awceptibie to suddat and wcxpected deterioration. For these reasons, chiral cohUnn!+in particular require carefill tnonitoring.
cleavage were optimized, and an RP-HPLC method that can directly
A number of SSTafor chirsl HPLC systana utilising a selc&on of common attd aovd elkI HPLC columns are preaawd. These columnsinclude various cycMutrin tmd protein phased.Common drug analytes have been chosen for these SSTs and include: propranolol; veraparnil;dprenolol; tioconazole, and benzoii.
trypsin and/or lysylendopeptidaseor cleaved with cyanogenbromide
analyze hydrophobic peptides dissolved in a sodium dodecylsulfate solutionwas developed. Reduced and S-carboxymethylatedTGP-943 was digested with
The resulting peptides were separated and fractionatedby RP-HPLC and then characterized by amino acid analysis. The resultssupported the amino acid compositionof each peptide expected from the cDNA sequence. Furthermore, it was confirmed that TGP-943 carries sugar
This work haa aigni6cant implicationsfor assuranceof robustnessin regulatory aubmisaiona
chains using digestionwith glycosidase,and the amino acid composition of the glycopeptidesrevealed that the sugar chains are mainly in the pre-S2 region.
FT-Raman spectroscopy has proved to be a useful technique for the study of the molecular basis of normal, healthy and abnormal, diseased human tissue’. Verrucae or warts are contagious, benign tumours resulting from a viral infection which affects only the skin, appearing as small papules or nodules on the skin or mucous membrane. Here we have used FT-Raman spectroscopy to study excised verrucae which had been successfully treated with a local application of a salicylic acid ointment. A liquid chromatography method for analysis of benzytpenicillin was examined in a daborative study. The method comprises an isocratii part, which is used in the assay. When the isocratic part is combined with gradient elution. the method is suiteble for purity control. The method prescribes the use of Cl8 stationary phase with 5 pm particle size and column dimensions of 25 x 0.46 cm i.d. a flow rate of 1.0 ml min-‘, an injector with a loop of about 20 pl and a UV detector set at 225 nm. Two mobile phases were used. Mobile phase A was a mixture of 0.5 M phosphate buffer pH 3.5~methanol-water (10:30:60. v/vhr) and mobile phase B was a mixture of the same components with a ratio (10:50:40. v/v/v). For the assay, isocratic elution was used with a mobile phase ratio A:B of 70:30. For the related substances test, isocratic elutiin combined with gradient elution was petformsd as follows: after isocratic elution of the benzylpenicillin peak with a mobile phase ratio A:8 used in the assay, a linear gradient elution was started to reach a mobile phase ratio A:B of 0:lOO over a period of 20 min. this ratio was held for 15 min. then the column was equilibrated with a mobile phase ratio A:B of 70:30 during 15 min. Five samples of benzylpenicillin (sodium and potassium salt) with varying purity were analysed. The main component and the impurities were determined. The results will be shown together with an anaiysii of variance and calculated estimates for the repeatability and reproducibilii of the method.
FT-Raman spectra were recorded using a Bruker FRA 106 Raman module mounted on a Bruker IFS 66 optical system, using NIR (1.064 urn) laser excitation. Spectra were typically recorded over 1000 scans at 4 cm“ at a laser power of 400-500 mW. Differences in the molecular structure of the stratum comeum across the surface of the sample were observed, and by comparison with normal and hyperkeratotic (callus) stratum wmeum it was concluded that the tissue around the edge of the sample was typically healthy skin. In the centre of the verruca, the molecular structure of the skin is altered and evidence for the presence of salicylic acid is observed. The salicylic acid is not observed in a pure crystalline state but appeared to be present wmplexed within the skin. No evidence for the presence of the other ointment components, lactic acid and flexible wllodion was found in the spectrum of the centre of the verruca This approach may help elucidate the molecular therapeutic agents acting on skin disorders.
basis for
‘Edwards, H.G.M. et al., J.Mol.Structure.. 347, 379 (1995)