Process for the isolation of a major harmful oxidant from cigarette smoke

A process for the isolation of p-benzosemiquinone of formula 1, a major harmful oxidant from cigarette smoke responsible for the oxidative damage of proteins and DNA, the said process further comprising

• (a) passing the whole cigarette smoke collected from conventional filter tipped cigarette having a tar content of 20-30 mg per cigarette into 30-60 mM potassium buffer at pH 7.4-7.8, filtering the above solution through 0.45 μm Millipore filter, adjusting the pH of the filtrate ranging between 7.4 to 7.6 by adding NaOH solution to obtain the desired cigarette smoke solution (cs solution);
• (b) extracting the above said cs solution thrice with equal volume of methylene chloride, discarding the lower methylene chloride layer and collecting the upper yellow coloured aqueous layer termed as aqueous extract of cigarette smoke;
• (c) extracting the above said aqueous layer of cigarette smoke twice with equal volume of water saturated n-butanol, lyophilizing the pooled yellow butanol extract in a Lyolab lyophilizer at a temperature ranging between −50°C. to −60° C. under vacuum followed by extraction of the lyophilized material twice with HPLC grade acetone and drying the acetone solution under vacuum and dissolving the said acetone extract with HPLC grade methanol;
• (d) subjecting the above said methanol solution to band TLC using non-fluorescent silica plates, developing the said silica plates using a mixture of toluene and ethyl acetate in a ratio of 80:20, taking out the plate and drying at about 25° C. to 30° C. using a drier, cutting small strips containing the developed material from both sides of the plates and keeping them in an iodine chamber for the location of the band corresponding to Rf 0.26, scraping the band and extracting the band material with HPLC grade acetone followed by collection of the acetone layer and drying it under vacuum;
• (e) dissolving the above said acetone extract which appeared as pale yellow needles by adding equal volume of milli Q water, extracting the aqueous solution with equal volume of HPLC grade water saturated n-butanol followed by drying the upper n-butanol layer in small glass tubes under vacuum to obtain the major cs oxidant with a purity of 98-99% and yield of 18-22 μg per cigarette;
• and
• (f) purifying the above said cs oxidant as obtained in step e by dissolving it in a mobile solvent comprising a mixture of methylene chloride and methanol in a ratio of 90:10(v/v) and injecting it in a HPLC instrument with a normal phase 25 cm silica column using a uv detector at 294 nm at a flow rate of 0.5 ml/min, at a temperature of about 25° C., at a pressure of about 29 kgf/cm2 and collecting the effluent which appears as a single peak at a retention time of 8.808 min with a purity of 100% and yield of 8.4% of the total cs oxidant present in the parent cs solution.

A process for the quantitative determination of p-benzosemiquinone of formula 1, a major harmful oxidant isolated from cigarette smoke responsible for the oxidative damage of proteins and DNA, the said process comprising the steps of

• (a) collecting tar or cs (cigarette smoke) solution from lighted conventional filtered tipped cigarettes,
• (b) collecting tar by lighting conventional filter-tipped cigarettes having a tar content of 20-30 mg per cigarette in a glass flask dipped in a mixture of ice and salt and allowing the tar to condense and settle at the bottom of the flask,
• (c) keeping the above said flask at room temperature and extracting the said tar with 30-60 mM potassium phosphate buffer at a pH ranging between 7.4 to 7.8, filtering the above solution through 0.45 μm Millipore filter and adjusting the pH of the filtrate ranging between 7.4 to 7.6 by adding NaOH solution to obtain the desired tar solution,
• (d) extracting the above said tar solution thrice with equal volume of methylene chloride, discarding the lower methylene chloride layer and collecting the upper yellow coloured aqueous layer termed as aqueous extract of cigarette smoke,
• (e) extracting the above said aqueous extract of cigarette smoke twice with equal volume of water saturated n-butanol, lyophilizing the pooled yellow butanol extract in a lyophilizer at a temperature ranging between −50° C. to −60° C. under vacuum followed by extraction of the lyophilized material twice with HPLC grade acetone and drying the acetone solution under vacuum and dissolving the said acetone extract with HPLC grade methanol,
• (f) subjecting the above said methanol solution to band TLC using non-fluorescent silica plates, developing the said silica plates using a mixture of toluene and ethyl acetate in a ratio of 80:20, taking out the said plate and drying it at about 25-30° C. using a drier followed by cutting small strips containing the developed material from both sides of the plates and keeping them in an iodine chamber for the location of the band corresponding to Rf 0.26, scraping the band and extracting the band material with HPLC grade acetone followed by collection of the acetone layer and drying it under vacuum,
• (g) dissolving the above said acetone extract which appeared as pale yellow needles by adding equal volume of milli Q water, extracting the resultant aqueous solution with equal volume of HPLC grade water saturated n-butanol followed by drying upper n-butanol layer in small glass tubes under vacuum to obtain the major cigarette smoke (cs) oxidant with a purity of 98-99% and yield of about 18-22 μg per cigarette, and
• (h) purifying the above said cs oxidant as obtained in step (g) by dissolving it in a mobile solvent comprising a mixture of methylene chloride and methanol in a ratio of 90:10 (v/v) and injecting it in a HPLC instrument with a normal phase 25 cm silica column using a uv detector at 294 nm at a flow rate of 0.5 ml/min, at a temperature of about 25° C. and at a pressure of about 29 kgf/cm2 followed by collecting the effluent which appears as a single peak at a retention time of 8.808 min with a purity of 100% and yield of 8.4% of the total cs oxidant present in the parent tar solution.

A process for the quantitative determination of p-benzosemiquinone of formula 1, a major harmful oxidant isolated from cigarette smoke responsible for the oxidative damage of proteins and DNA, the said process further comprising

• (a) passing the whole cigarette smoke collected from conventional filter tipped cigarette having a tar content of 20-30 mg per cigarette into 30-60 mM potassium buffer at pH 7.4-7.8, filtering the above solution through 0.45 μm Millipore filter, adjusting the pH of the filtrate ranging between 7.4 to 7.6 by adding NaOH solution to obtain the desired cigarette smoke solution (cs solution),
• (b) extracting the above said cs solution thrice with equal volume of methylene chloride, discarding the lower methylene chloride layer and collecting the upper yellow colored aqueous layer termed as aqueous extract of cigarette smoke,
• (c) extracting the above said aqueous layer of cigarette smoke twice with equal volume of water saturated n-butanol, lyophilizing the pooled yellow butanol extract in a Lyolab lyophilizer at a temperature ranging between −50° C. to −60° C. under vacuum followed by extraction of the lyophilized material twice with HPLC grade acetone and drying the acetone solution under vacuum and dissolving the said acetone extract with HPLC grade methanol,
• (d) subjecting the above said methanol solution to band TLC using non-fluorescent silica plates, developing the said silica plates using a mixture of toluene and ethyl acetate in a ratio of 80:20, taking out the plate and drying at about 25° C. to 30° C. using a drier, cutting small strips containing the developed material from both sides of the plates and keeping them in an iodine chamber for the location of the band corresponding to Rf 0.26, scraping the band and extracting the band material with HPLC grade acetone followed by collection of the acetone layer and drying it under vacuum,
• (e) dissolving the above said acetone extract which appeared as pale yellow needles by adding equal volume of milli Q water, extracting the aqueous solution with equal volume of HPLC grade water saturated n-butanol followed by drying the upper n-butanol layer in small glass tubes under vacuum to obtain the major cs oxidant with a purity of 98-99% and yield of 18-22 μg per cigarette, and
• (f) purifying the above said cs oxidant as obtained in step e by dissolving it in a mobile solvent comprising a mixture of methylene chloride and methanol in a ratio of 90:10(v/v) and injecting it in a HPLC instrument with a normal phase 25 cm silica column using a uv detector at 294 nm at a flow rate of 0.5 ml/min, at a temperature of about 25° C., at a pressure of about 29 kgf/cm2 and collecting the effluent which appears as a single peak at a retention time of 8.808 min with a purity of 100% and yield of 8.4% of the total cs oxidant present in the parent cs solution.