[Show abstract][Hide abstract] ABSTRACT: Electronic cigarettes (e-cigarettes) are advertised as being safer than tobacco cigarettes products as the chemical compounds inhaled from e-cigarettes are believed to be fewer and less toxic than those from tobacco cigarettes. Therefore, continuous careful monitoring and risk management of e-cigarettes should be implemented, with the aim of protecting and promoting public health worldwide. Moreover, basic scientific data are required for the regulation of e-cigarette. To date, there have been reports of many hazardous chemical compounds generated from e-cigarettes, particularly carbonyl compounds such as formaldehyde, acetaldehyde, acrolein, and glyoxal, which are often found in e-cigarette aerosols. These carbonyl compounds are incidentally generated by the oxidation of e-liquid (liquid in e-cigarette; glycerol and glycols) when the liquid comes in contact with the heated nichrome wire. The compositions and concentrations of these compounds vary depending on the type of e-liquid and the battery voltage. In some cases, extremely high concentrations of these carbonyl compounds are generated, and may contribute to various health effects. Suppliers, risk management organizations, and users of e-cigarettes should be aware of this phenomenon.
International journal of environmental research and public health. 01/2014; 11(11):11192-11200.
[Show abstract][Hide abstract] ABSTRACT: Objective: To determine constituents of fillers and mainstream smoke from Neo Cedar. Methods: Neo Cedar is a second-class over-the-counter (OTC) drug and similar to cigarettes in a number of ways. In particular, the design and usage are very similar to those of cigarettes. For the fillers of the drug, the levels of nicotine, tobacco-specific nitrosamines (TSNA), and heavy metals, and mutagenicity were determined using the methods for cigarette products. For the mainstream smoke, the levels of tar, nicotine, carbon monoxide (CO), TSNA, polycyclic aromatic hydrocarbons (PAH), and carbonyl compounds were also determined using the methods for cigarettes. The mainstream smoke from the drug were collected with a smoking machine using two smoking protocols (ISO and Health Canada Intense methods). Results: The nicotine and total TSNA levels in the fillers of the drug averaged 2.86 mg/g and 185 ng/g, respectively. The nine species of heavy metals were also detected in the fillers of the drug. The levels of nicotine, tar, CO, TSNA, PAH, and carbonyl compounds of mainstream smoke from the drug were higher when determined using the HCI regime than when using the ISO regime. The mutagenicity of the mainstream smoke determined using the HCI regime was also higher than that determined using the ISO regime. Conclusion: In this study, all constituents of Neo Cedar were determined by methods for cigarette products. The drug had a ventilation hole on its filter. Thus, its constituents are different from those determined by the smoking protocols. Neo Cedar users should be careful of higher exposure to the hazardous gases owing to smoking patterns.
Nippon Eiseigaku Zasshi (Japanese Journal of Hygiene) 01/2014; 69(1):31-8.
[Show abstract][Hide abstract] ABSTRACT: The tobacco tax in Japan was increased in October, 2010. Subsequently, the quantity of sales of domestic cigarette products decreased temporarily. However, smokers could import cigarettes directly through the internet. Because of the high value of the yen and the low price of foreign cigarettes, an individual could import cigarettes easily; however, the contents and emissions from privately imported cigarettes have not been investigated yet. In this study, we carried out a comparative analysis of domestic and privately imported cigarettes.
Nihon eiseigaku zasshi. Japanese journal of hygiene. 01/2014; 69(3):205-10.
[Show abstract][Hide abstract] ABSTRACT: We developed a simple method for the simultaneous determination of volatile organic compounds (VOCs) and carbonyls in the mainstream cigarette smoke using a sorbent cartridge at ambient temperature without the traditional cryogenic impinger. A sorbent cartridge is installed between intake filter and the pump of the smoking machine. Collection of cigarette mainstream smoke is performed according to the Canadian Intense regime or the ISO regime. As adsorbent, Carboxen 572 (CX-572) is the most suitable for collection of VOCs and carbonyls in the mainstream cigarette smoke. Elution of VOCs and carbonyls from CX-572 is performed by the two-step elution with carbon disulfide and methanol. VOCs are eluted by first elution with carbon disulfide and carbonyls are eluted by second elution with methanol. For VOCs, a portion of eluate is analyzed by gas chromatography-mass spectrometry. For carbonyls, a portion of eluate is derivatized with enriched 2,4-dinitrophenylhydrazine solution and analyzed by high-performance liquid chromatography. Measurement values by CX-572 cartridge method are very close to those obtained by traditional impinger method except for 2-butanone. Impinger methods use 2,4-dinitrophenylhydrazine solution containing 50% water and 2-butanone-DNPhydrazone may be hydrolyzed with water. In the CX-572 method, the hydrolysis of 2-butanone is prevented because the eluate solution contains no water. CX-572 method can measure cigarette smoke resulting from not only one whole cigarette but also from one puff volume because of its high sensitivity and simple operation.
Journal of Chromatography A 09/2013; · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Carbonyl compounds in E-cigarette smoke mist were measured using coupled silica cartridges impregnated with hydroquinone and 2,4-dinitrophenylhydrazine, followed by high-performance liquid chromatography. A total of 363 E-cigarettes (13 brands) were examined. Four of the 13 E-cigarette brands did not generate any carbonyl compounds, while the other nine E-cigarette brands generated various carbonyl compounds. However, the carbonyl concentrations of the E-cigarette products did not show typical distributions, and the mean values were largely different from the median values. It was elucidated that E-cigarettes incidentally generate high concentrations of carbonyl compounds.
[Show abstract][Hide abstract] ABSTRACT: Objective: To measure the nicotine and tobacco-specific nitrosamine (TSNA) contents in whole tobacco from a variety of Japanese domestic cigarette brands and to determine mutagencity. Methods: The test cigarettes were the top ten best selling cigarette brands in Japan in 2006. The nicotine content in whole tobacco was measured by a modified version of the CORESTA Recommended method. The level of TSNA was measured by a modified version of the Health Canada method. Mutagenicity was assessed using the Salmonella typhimurium strains TA100, TA98 and YG1024 with the metabolic activation system (S9mix) by preincubation assays. Results: The nicotine content in the test cigarettes averaged 15.7±1.2 mg/g, and was in the range between 13.7 and 17.2 mg/g. The level of TSNA averaged 1,750 ng/g, and was in the range between 931 and 2,490 ng/g. Mutagenicity was pseudopositive in several samples of the YG1024 tester strain with and without S9mix. Conclusion: The cigarettes brands were categorized into four groups (Ultra-low, Low, Medium, and High) based on the nominal nicotine yield figures printed on the cigarette packets. The nicotine content in whole tobacco of the High group was the highest. However, the level of TSNA of the high group was the lowest. The analyses of hazardous chemical compounds in whole tobacco can contribute to the reduction and regulation of the toxicity of tobacco products.
Nippon Eiseigaku Zasshi (Japanese Journal of Hygiene) 01/2013; 68(1):46-52.
[Show abstract][Hide abstract] ABSTRACT: A typical method for the measurement of glutaraldehyde (GLA) employs 2,4-dinitrophenylhydrazine (DNPH) to form GLA-DNPhydrazone derivatives. However, this method is subject to analytical errors because GLA-DNPhydrazone is a quaternary bis-derivative and forms three geometric isomers (E-E, E-Z and Z-Z) as a result of the two C[double bond, length as m-dash]N double bonds. To overcome this issue, a method for transforming the C[double bond, length as m-dash]N double bond into a C-N single bond, using reductive amination of DNPhydrazone derivatives, has been applied. The amination reaction of GLA-DNPhydrazones with 2-picoline borane is accelerated with catalytic amounts of acid and is completed within 10 minutes in the presence of 100 mmol L(-1) phosphoric acid. Reduction of GLA-DNPhydrazone by 2-picoline borane is unique and results in the formation of N-(2,4-dinitrophenyl)-1-piperidinamine (DNPPA). NMR and LC-APCI-MS data confirmed the product identification. DNPPA is very stable and did not change when stored for at least four weeks at room temperature. DNPPA has excellent solubility of 14.6 g L(-1) at 20 °C in acetonitrile. The absorption maximum wavelength and the molar absorptivity of DNPPA were 351 nm and 4.2 × 10(4) L mol(-1) cm(-1) respectively. Complete separation between the reduced forms of C1-C10 aldehyde DNPhydrazones, including DNPPA, can be achieved by operating the reversed-phase high-performance liquid chromatograph at 351 nm in gradient mode using a C18 amide column. The reductive amination method for GLA overcomes analytical errors caused by E-E, E-Z and Z-Z geometrical isomers.
The Analyst 07/2012; 137(18):4274-9. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES: Although the relative risk of lung cancer due to smoking is reported to be lower in Japan than in other countries, few studies have examined the characteristics of Japanese cigarettes or potential differences in smoking patterns among Japanese smokers. METHODS: To examine tar, nicotine and carbon monoxide (TNCO) emissions from ten leading cigarettes in Japan, machine smoking tests were conducted using the International Organization for Standardization (ISO) protocol and the Health Canada Intense (HCI) protocol. Smoking topography and tobacco-related biomarkers were collected from 101 Japanese smokers to examine measures of exposure. RESULTS: The findings indicate considerable variability in the smoking behavior of Japanese smokers. On average, puffing behaviors observed among smokers were more similar to the parameters of the HCI protocol, and brands with greater ventilation that yielded lower machine values using the ISO protocol were smoked more intensely than brands with lower levels of ventilation. The smokers of "ultra-low/low" nicotine-yield cigarettes smoked 2.7-fold more intensively than those of "medium/high" nicotine-yield cigarette smokers to achieve the same level of salivary cotinine (p = 0.024). CO levels in expiratory breath samples were associated with puff volume and self-reported smoking intensity, but not with nominal values of nicotine-yield reported on cigarette packages. CONCLUSIONS: Japanese smokers engaged in "compensatory smoking" to achieve their desired nicotine intake, and levels of exposure were greater than those suggested by the nominal value of nicotine and tar yields reported on cigarette packages.
Environmental Health and Preventive Medicine 07/2012;
[Show abstract][Hide abstract] ABSTRACT: A diffusive sampling device, the DSD-NH3, has been developed for measurement of ammonia in air. The DSD-NH3 comprises silica gel impregnated with phosphoric acid as the absorbent, a porous sintered polyethylene tube that acts as a diffusive membrane, and a small polypropylene syringe that is used for the elution of analytes from the absorbent. Silica gel impregnated with phosphoric acid is used as absorbent for the DSD-NH3; basic gases in ambient air, including ammonia, are trapped in the DSD-NH3 device by their reaction with phosphoric acid in the sampler to form their corresponding phosphoric acid salts. After collection, the DSD-NH3 samplers are eluted by water. Cations in the eluate, including ammonium ions, are analyzed by ion chromatography. A side-by-side comparison was made with active samplers, demonstrating good correlation (r2 = 0.996). The sampling rate (94.5 ml min−1) was determined from comparison with an active sampling method and sampling rates. The sampling rate is also calculated from the respective molecular weights according to a rule based on Graham’s law. The theoretical sampling rate with the DSD-NH3 is 95.4 ml min−1 and agrees with the experimental value (94.5 ml min−1). Little influence of wind velocity on the sampler was observed. The relative standard deviations for ammonia concentrations were 4.7% with face velocity ranging 0–5.0 m/s.
[Show abstract][Hide abstract] ABSTRACT: Mainstream smoke from cigarettes contains tobacco-specific N'-nitrosamines (TSNAs) listed as Group 1 and 3 carcinogens by the International Agency for Research on Cancer (IARC). Herein, we report on a method of measuring the concentrations of TSNAs in mainstream smoke from the ten top-selling Japanese cigarette brands using an ISO regime by International Organization for Standardization (ISO) and HCI regime of Health Canada.
Tar in mainstream smoke was collected on a Cambridge filter pad using a smoking machine. The filter pad was immersed in 40 mL of ammonium acetate (pH 6.8) and shaken for 30 min. The extract was then loaded into a C18 column. After washing with 5 mL of 10% methanol and eluting with 5 mL of 70% methanol, the eluate was concentrated to 1 mL for LC-MS/MS analysis.
The concentrations of TSNAs in all cigarette brands were higher when determined using the HCI regime than when determined using the ISO regime. Furthermore, the concentrations of TSNAs measured using both the ISO and HCI regimes showed negligible correlation to the tar and nicotine concentrations indicated on package labels. The cigarette samples used in the study were categorized into four classes: ultralow-, low-, medium-, and high-yield brands, which corresponded to 1, 3-6, 8-10, and 14 mg tar/cigarette, respectively. The concentration of TSNAs in ultralow-yield cigarettes was 210 ng/cigarette, as measured using the HCI regime, which was nearly equal to that in high-yield cigarettes (180 ng/cigarette).
Exposure to TSNAs from mainstream smoke from ultralow-yield cigarettes is comparable to that from high-yield cigarettes. To properly evaluate the risk of smoking, not only the concentrations of tar and nicotine but also those of other chemicals, including TSNAs, should be printed on package labels.
Nippon Eiseigaku Zasshi (Japanese Journal of Hygiene) 05/2012; 67(3):423-30.
[Show abstract][Hide abstract] ABSTRACT: The most widely used method for measuring carbonyl compounds in air is 2,4-dinitrophenylhydrazine (DNPH) derivatization followed by high-performance liquid chromatography (HPLC). However, substantial negative interference caused by the presence of ozone in air has been reported. To avoid the influences of ozone, a potassium iodide scrubber (KI-scrubber) is commonly used. However, when air sampling using a DNPH-cartridge and a KI-scrubber is performed under conditions of high humidity, moist potassium iodide in the KI-scrubber traps carbonyls before they reach the DNPH-cartridge. Moreover, wet KI reacts with I(2) to form KI(3) and this oxidative reagent moves to the DNPH-cartridge and destroys the DNPH and DNPhydrazone derivatives. In order to alleviate these problems, new ozone scrubbers (BPE-scrubber, HQ-scrubber) have been developed. BPE-scrubber and HQ-scrubber consist of silica gel particles impregnated with trans-1,2-bis-(2-pyridyl) ethylene (BPE) and hydroquinone (HQ), respectively. BPE reacts with ozone to form pyridine aldehyde and HQ reacts with ozone to form benzoquinone. The amounts of reducing agent in silica gel (130 mg) for ozone scrubber are 1% (w/w) for BPE-cartridge; 0.2% (w/w) for HQ-scrubber. These scrubbers can be used in air containing 140 μg/m(3) of ozone for 24h at a flow rate of 200 mL/min. When the relative humidity exceeded 80%, KI in the KI-scrubber was gradually moistened and changed to yellow in color. Peak abundance of formaldehyde, acetaldehyde and acetone DNPhydrazones was diminished to 25%, 15%, and 2%, respectively, compared with the BPE-scrubber or HQ-scrubber. When using a BPE-scrubber or HQ-scrubber, decomposition of DNPH and DNPhydrazones was not observed at a wide range of relative humidities (3-97%).
Journal of Chromatography A 03/2012; 1229:293-7. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new diffusive sampling method for the simultaneous determination of ozone and carbonyls in air has been developed. In this method, silica gel impregnated with a mixture of trans-1,2-bis(2-pyridyl)ethylene (2BPE) and 2,4-dinitrophenylhydrazine (DNPH) is used as the absorbent; further, a porous sintered polyethylene tube (PSP-diffusion filter), which acts as a diffusive membrane, and a small polypropylene syringe (PP-reservoir) for elution of the analytes from the absorbent are used. The carbonyls present in air react with DNPH in the absorbent to form hydrazone derivatives. Concurrently, ozone in the air reacts with 2BPE to form pyridine-2-aldehyde, which immediately reacts with DNPH to form a pyridine-2-aldehyde hydrazone derivative. All the hydrazones derived from airborne carbonyls, including pyridine-2-aldehyde (formed from ozone), are completely separated and analyzed by high-performance liquid chromatography. The sampling rates of ozone (44.6 mL min(-1)) and formaldehyde (72.0 mL min(-1)) are determined by comparison with the rates obtained in an active sampling method. The sampling rates of other carbonyl compounds are calculated from the respective molecular weights according to a rule based on Graham's law. The calculated sampling rates agree with the experimental values. The DSD-BPE/DNPH method is advantageous because it is simple and allows for the simultaneous analysis of ozone and carbonyls.
[Show abstract][Hide abstract] ABSTRACT: Derivatization of carbonyl compounds with 2,4-dinitrophenylhydrazine (DNPH) is one of the most widely used analytical methods. In this article, we highlight recent advances using DNPH provided by our studies over past seven years. DNPH reacts with carbonyls to form corresponding stable 2,4-DNPhydrazone derivatives (DNPhydrazones). This method may result in analytical error because DNPhydrazones have both E- and Z-stereoisomers caused by the CN double bond. Purified aldehyde-2,4-DNPhydrazone demonstrated only the E-isomer, but under UV irradiation and the addition of acid, both E- and Z-isomers were seen. In order to resolve the isometric problem, a method for transforming the CN double bond of carbonyl-2,4-DNPhydrazone into a C-N single bond, by reductive amination using 2-picoline borane, has been developed. The amination reactions of C1-C10 aldehyde DNPhydrazones are completely converted into the reduced forms and can be analyzed with high-performance liquid chromatography. As a new application using DNPH derivatization, the simultaneous measurement of carbonyls with carboxylic acids or ozone is described in this review.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 10/2010; 879(17-18):1282-9. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are many gaseous air pollutants found in indoor air. It is very important to precisely measure the concentration of these compounds in order to evaluate the risk to human health and to reduce their concentrations. A diffusive sampling device is suitable for measurement of indoor air, because these are small, light, and can be used without a power supply for the pump. In this study, representative gaseous air pollutants in winter indoor and outdoor air were measured using diffusive sampling devices. Furthermore, the relationship between gaseous air pollutants, secondary formation mechanism, and the outbreak source were examined. The indoor concentrations of aldehydes, nitrogen dioxide and ammonia were higher than outdoor concentrations. By contrast, indoor concentrations of ozone were lower than outdoor concentrations. The indoor concentrations of nitrogen dioxide in 43% houses exceeded the maximum limit stated by environmental law (60 ppb). It was suggested that the main emission sources of nitrogen dioxide are kerosene and gas stoves. In addition, it was suggested that carbonyl compounds are formed by interactions between volatile organic compounds (VOCs) and ozone from outdoor air. Formic acid was estimated to be formed by the oxidation of formaldehyde with ozone, because a positive correlation between formaldehyde and formic acid, and an inverse correlation between formaldehyde and ozone, were observed in indoor air.
[Show abstract][Hide abstract] ABSTRACT: A new method for the determination of acrolein and other carbonyls in cigarette smoke using a dual cartridge system has been developed. Each cartridge consists of reagent-impregnated silica particles. The first contains hydroquinone (HQ) for the inhibition of acrolein polymerization, while the second contains 2,4-dinitrophenylhydrazine (DNPH) for the derivatization of carbonyls. Smoke samples were firstly drawn through the cartridge containing HQ-impregnated silica (HQ-silica) and then through the DNPH-impregnated silica (DNPH-silica). Acrolein in the sample was completely trapped in the first HQ-silica cartridge. Some other airborne carbonyls were also trapped by the HQ-silica, and those that pass through were trapped in the second DNPH-silica cartridge. Extraction was performed in the reverse direction to air sampling. When solvent was eluted through the dual-cartridges, excess DNPH was washed into the HQ bed where it reacted with acrolein and other trapped carbonyls to form the corresponding hydrazone derivatives. All of the hydrazones derived from airborne carbonyls were completely separated and measured using high-performance liquid chromatography. This HQ-DNPH-method can be applied for the determination of acrolein and other alpha,beta-unsaturated aldehydes, such as crotonaldehyde, in cigarette smoke.
Journal of Chromatography A 06/2010; 1217(26):4383-8. · 4.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously developed a new method using trans-1,2-bis-(4-pyridyl)ethylene (4-BPE) and 2,4-dinitrophenylhydrazine (DNPH) for the simultaneous determination of ozone and carbonyls in air using a two-bed cartridge system. In this method, 4-BPE was used to capture ozone. However, the method suffered from long reaction times in the eluate, low solubility of the DNPH derivative, and a strong dependence on atmospheric moisture. These problems could be overcome by using trans-1,2-bis-(2-pyridyl)ethylene (2-BPE) in place of 4-BPE. The efficiency of the reaction of ozone with 2-BPE to form pyridine-2-aldehyde (2PA) is higher than the corresponding reaction with 4-BPE. Under the optimized elution conditions, the reaction times of 2PA and 4PA with DNPH are within 15 and 120 min, respectively. In the elution from the sampling cartridge, 2PA formed from 2-BPE and ozone is easier to dissolve in the elution solvent. A stronger influence of humidity was observed in ozone recovery by the 4-BPE/DNPH method. 2-BPE exhibits a maximum reaction efficiency of 84% at 32% relative humidity (RH), while 4-BPE attains a maximum reaction efficiency of 82% at 49% RH. Humidity has much less influence on the reaction of 2-BPE with ozone. Above 18% RH, the reaction efficiency of 2-BPE with ozone is in the range 80-84%. Thus, 2-BPE is the more useful reagent for ozone analysis. The concentrations of ozone and carbonyls by the improved 2-BPE/DNPH method corresponded with the mean value by an ozone auto analyzer in an air monitoring station and a DNPH cartridge coupled with a KI-ozone scrubbing cartridge.
[Show abstract][Hide abstract] ABSTRACT: Based upon the Framework Convention on Tobacco Control (FCTC), the World Health Organization (WHO) ha sr ecommended that health authorities disclose toxicological properties of cigarette mainstream smoke (MSS) obtained not only according to US Federal Trade Commission (FTC)/International Organization for Standardization (ISO) conditions but also by more intense conditions such as the Health Canada Intense (HCI) condition. This is because smokers are believed to smoke more intensely than machine smoking under the ISO regimen. Because there are no previous reports on the toxicological properties of MSS of Japanese cigarettes under the HCI condition, we determined nicotine and water contents by gas chromatography (GC-MS and GC/Thermal Conductivity Detector (TCD)) for three product lots each of the ten bestselling brands of Japanese cigarettes following the WHO protocol. One of the three lots of each MSS condensate was also resolved in dimethylsulfoxide and investigated by Ames preincubation assay using Salmonella typhimurium TA100, TA98, and YG1024 strains with and without metabolic activation (rat liver S9 mix). Nicotine and tar yields with the HCI regimen were higher than those with ISO, the latter being very close to the values described on the packages of each cigarette brand. Mutagenicity was mainly observed in TA98 and YG1024 with metabolic activation. Mutagenic activity of MSS with the HCI regimen was 1.4-9 times higher than that with the ISO regimen. Based on YG1024 with activation, high activities were observed in several "low yield" brands. The activity of "low-yield" brands with the HCI regimen was not always lower than that of regular-yield brands with the ISO regimen. These results suggest that "low yield" cigarettes do not result in reduced exposure or reduced risk to humans.
Journal of Health Science - J HEALTH SCI. 01/2009; 55(3):421-427.
[Show abstract][Hide abstract] ABSTRACT: A new method for the determination of carbonyls in air using 2,4-dinitrophenylhydrazine (DNPH) has been developed. The traditional method for the measurement of carbonyl compounds, using DNPH to form the corresponding 2,4-dinitrophenylhydrazone (DNPhydrazone) derivatives, is subject to analytical errors because DNPhydrazones form both E- and Z-geometrical isomers as a result of the C=N double bond. To overcome this issue, a method for transforming the C=N double bond into a C-N single bond, using reductive amination of DNPhydrazone derivatives, has been developed. Reductive amination of aldehyde DNPhydrazones was carried out by adding 2-picoline borane acetonitrile solution in eluate through the DNPH-cartridge. The amination reactions of C(1)-C(10) aldehyde DNPhydrazones were completely converted into the reduced forms within 40 min in the presence of 1 mmol/L 2-picoline borane and 20 mmol/L of phosphoric acid. These reduced forms were very stable and did not change when stored for 2 weeks at room temperature. The absorption maximum wavelengths of the reduced forms from C(1)-C(10) aldehyde DNPhydrazones were 351-352 nm and shifted 6-7 nm toward shorter wavelengths when compared to the corresponding DNPhydrazones, and the molar absorption coefficients were 1.5 x 10(4) (C(1)) to 2.2 x 10(4) L/mol/cm (C(10)). Complete separation between C(1)-C(10) aldehyde DNPhydrazones and the corresponding reduced forms can be achieved by operating the HPLC in gradient mode using an Ascentis RP-Amide column (150 mm x 4.6 mm i.d.). The RSDs of DNPhydrazone (Z + E) peak areas ranged from 0.40-0.66 and those of the corresponding reduced forms ranged from 0.26-0.41. It was shown that the reductive amination method gave improved HPLC analytical precision because of the absence of isomers.