Determination of theobromine, theophylline, and caffeine in by-products of cupuacu and cacao seeds by high-performance liquid chromatography

Theobromine, theophylline, and caffeine are determined simultaneously by a rapid and selective reversed-phase high-performance liquid chromatography (HPLC) method with UV detection in by-products of cupuacu and cacao seeds. The determination is carried out in the raw and roasted ground cupuacu seeds and in the corresponding powders obtained after pressure treatment. The by-products of both cupuacu seeds and cacao seeds are obtained under the same technological conditions. The HPLC method uses isocratic elution with a mobile phase of methanol-water-acetic acid (80:19:1) (v/v) at a flow rate of 1 mL/min and UV absorbance detection at 275 nm. Total elution time for these analytes is less than 10 min, and the detection limit for all analytes is 0.1 mg/g. The amounts of theobromine and caffeine found in all the cupuacu samples are one or more orders of magnitude lower than those from cacao. Theophylline is found in all cacao samples except for the roasted ground paste, and it is only found in the roasted ground paste in the cupuacu samples.     

高效液相色谱法测定乳制品中的双氰胺

建立了乳制品中双氰胺的高效液相色谱(HPLC)检测方法。样品经乙腈提取后采用HPLC测定。优化后的色谱条件:XBridge Amide柱(250 mm×4.6 mm, 3.5 μm),流动相为乙腈和水(体积比为90:10,含0.2%甲酸),流速为1.0 mL/min,检测波长为218 nm。方法在0.5~50 mg/L范围内线性关系良好,相关系数(r²)为0.9999,回收率在96.7%~101.0%之间,RSD为4.5%~4.9%(n=6),检出限为0.2 mg/kg(S/N=3),定量限为0.5 mg/kg(S/N=10)。该方法简单、准确、灵敏度高,适用于各种乳制品中双氰胺的检测。     

Determination of caffeine and associated compounds in food, beverages, natural products, pharmaceuticals, and cosmetics by micellar electrokinetic capillary chromatography

A method is described for quantitating caffeine, theobromine, theophylline, paracetamol, propyphenazone, acetylsalicylic acid, salicylic acid, and codeine phosphate in corresponding real samples of food, beverages, natural products, pharmaceuticals, and cosmetic preparations by micellar electrokinetic capillary chromatography. The separation is carried out at 25 degrees C and 25 kV, using a 20mM phosphate buffer (pH 9.0), 80mM sodium dodecyl sulfate, and 7.5% (v/v) acetonitrile. UV detection is at 210 nm. The method is shown to be specific, accurate (recoveries over the range 98.9-101.2%), linear over the tested range (correlation coefficients >/= 0.9993), and precise (relative standard deviation below 2.1%). The method is applied for the quantitative analysis of these compounds in different foods, beverages, natural products, pharmaceuticals, and cosmetic products.     

Determination of adenine,caffeine, theophylline and theobromine by HPLC with amperometric detection

A relatively simple method for quantifying caffeine, theobromine, theophylline and adenine by HPLC with amperometric detection was developed. A C₁₈-column and an isocratic elution with phosphate buffer pH 3.5/methanol (90 : 10) were employed for the chromatographic separation of the investigated compounds. The optimal detection potential was +1.4 V. The limits of detection were 0.4 ng for adenine, 1 ng for theophylline and 2.5 ng for caffeine and theobromine. The method was applied to the determination of these purine alkaloids in beverages, tea, coffee and cacao. The determination was carried out directly or after solid-phase extraction.     

Application of liquid chromatography to the simultaneous determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine, and thiamine in pharmaceutical preparations.

This paper describes a rapid reversed-phase liquid chromatographic method, with UV detection, for the simultaneous determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine, and thiamine in pharmaceutical preparations. A reversed-phase C18 Nucleosil column is used. The mobile phase consists of 2 successive eluants: water (5 min) and acetonitrile-water (75 + 25, v/v; 9 min), both adjusted to pH 2.1 with phosphoric acid. Before determination acetylsalicylic acid is completely converted to salicylic acid by alkaline hydrolysis. Salicylic acid, caffeine, paracetamol, pyridoxine, and thiamine are all detected at 285 nm, whereas codeine is detected at 240 nm. Calibration curves were linear for salicylic acid, caffeine, paracetamol, and pyridoxine in the range of 50-500 mg/L, and for codeine and thiamine in the range of 50-1000 mg/L. The method was applied to the analysis of 13 fortified commercial pharmaceutical preparations. Recoveries ranged from 92.6 to 105.5%, with relative standard deviations of 1.1-5.8%.     

High performance liquid chromatographic determination of methylxanthines in canine serum, gastric and pancreatic juices

A convenient high performance liquid chromatographic method for the determination of methylxanthines in biological samples is described. Separation was achieved by reversed phase chromatography using a mobile phase consisting of tetrahydrofuran + methanol + 0.01M potassium dihydrogen phosphate, pH 3.5 (1:20:79, v/v/v), on a 7 microns C18 column and a C18 Lichrosorb precolumn at a flow rate of 0.8 mL/min. Levels varying from 0.25-16 mg/L could be detected by UV at 280 nm. In this range, standard curves were established for 4 methylxanthines: theobromine, paraxanthine, theophylline and caffeine in 4 media: mobile phase, serum, gastric and pancreatic juices, and were found to be linear (r greater than or equal to 0.9975). Overall characteristics of the method were determined as: percent recovery (89.54%), accuracy (greater than or equal to 99.4%) and reproducibility (greater than or equal to 95%). Retention times ranged from 4.21 +/- 0.01 (1-methyluric acid) to 10.8 +/- 0.03 min (caffeine). Animal experiments (5 and 10 mg/kg boluses) were used to determine caffeine half life in dog's blood (310 +/- 46 and 453 +/- 59 min, respectively) and its secretion into pentagastrin stimulated gastric juice (mean concentrations 2.51 and 6.04 mg/L; mean outputs 351 and 1206 micrograms/2.25 h; both statistically different at p less than 0.001 level).     

Separation of caffeine and theophylline in poly(dimethylsiloxane) microchannel electrophoresis with electrochemical detection

A method for the rapid separation and sensitive determination of caffeine and theophylline was presented in poly(dimethylsiloxane) (PDMS) microchannel electrophoresis integrated with electrochemical detection. By using methanol as an additive, the peak shape and resolution were essentially improved. The analytes were well separated within only 40s in the running buffer of 5.0mM borate solution (pH 9.2) containing 10% (v/v) methanol. The linear ranges were from 6microM to 0.6mM and the detection limits were 4microM for caffeine and theophylline, respectively. The proposed method has been successfully applied to determine caffeine and theophylline in rat serum and urine.     

高效液相色谱法同时测定功能饮料中烟酰胺、咖啡因、维生素B_6、柠檬黄、胭脂红和苯甲酸的含量

建立了一种同时快速检测功能饮料中烟酰胺、咖啡因、维生素B_6、柠檬黄、胭脂红和苯甲酸6种食品添加剂的高效液相色谱方法。6种食品添加剂的检出限分别为烟酰胺0.1 mg/L,咖啡因0.1 mg/L,维生素B6 0.2 mg/L,柠檬黄0.2 mg/L,胭脂红0.1 mg/L,苯甲酸0.1 mg/L,测定结果的相对标准偏差不大于4.57%(n=6),加标回收率在95.80%~113.68%之间。该方法满足GB/T 5009.197–2003,GB/T 23495–2009和SN/T 2105–2008对于上述6种食品添加剂检出限的要求。     

Simultaneous determination of caffeine, theobromine, and theophylline by high-performance liquid chromatography

This work relates the development of an analytical methodology to simultaneously determine three methylxanthines (caffeine, theobromine, and theophylline) in beverages and urine samples based on reversed-phase high-performance liquid chromatography. Separation is made with a Bondesil C18 column using methanol-water-acetic acid or ethanol-water-acetic acid (20:75:5, v/v/v) as the mobile phase at 0.7 mL/min. Identification is made by absorbance detection at 273 nm. Under optimized conditions, the detection limit of the HPLC method is 0.1 pg/mL for all three methylxanthines. This method is applied to urine and to 25 different beverage samples, which included coffee, tea, chocolate, and coconut water. The concentration ranges determined in the beverages and urine are: < 0.1 pg/mL to 350 microg/mL and 3.21 microg/mL to 71.2 microg/mL for caffeine; < 0.1 pg/mL to 32 microg mL and < 0.1 pg/mL to 13.2 microg/mL for theobromine; < 0.1 pg/mL to 47 microg/mL and < 0.1 pg/mL to 66.3 microg/mL for theophylline. The method proposed in this study is rapid and suitable for the simultaneous quantitation of methylxanthines in beverages and human urine samples and requires no extraction step or derivatization.     

高效液相色谱法同时测定制药废水中的交沙霉素、茶碱及扑热息痛

建立了同时测定制药废水中残留的交沙霉素、茶碱、扑热息痛等3种药物的高效液相色谱方法。样品经固相萃取处 理后进行色谱分析。采用的色谱条件:色谱柱为Hypersil ODS柱（4.6 mm i.d.×200 mm）;流动相A液为0.025 mol/L KH2PO4-H3PO4 缓冲液(pH 2.75),流动相B液为甲醇;梯度洗脱;紫外检测波长为230 nm(交沙霉素)、272 nm(茶碱)、243 nm(扑热息痛)。制药废水中3种药物的加标回收率均高于93%,相对标准偏差(n=6)小于2.1%,检测下限(S/N=3)不高于1.0 μg/L。该方法已应用于制药废水的生物强化降解研究。     

Optimization and validation of a method for the determination of caffeine, 8-chlorotheophylline and diphenhydramine by isocratic high-performance liquid chromatography. Stress test for stability evaluation

The optimization of a HPLC method for caffeine, 8-chlorotheophylline and diphenhydramine separation with UV detection at 229 nm is described. The conditions studied included: stationary phase, compositions of mobile phases with pH modulators. Optimal conditions were: SymmetryShield RP8 column and acetonitrile–(0.01 M H₃PO₄–triethylamine, pH 2.8) (22:78, v/v). Validation was performed using standards and a pharmaceutical preparation containing the compounds described above. Results from both standards and samples show suitable validation parameters. The pharmaceutical grade substances were tested by factors that could influence the chemical stability. These reaction mixtures were analyzed to evaluate the capability of the method to separate degradation products. Degradation products did not interfere with the determination of the substances tested by the assay.     

HPLC, TLC, and first-derivative spectrophotometry stability-indicating methods for the determination of tropisetron in the presence of its acid degradates

Three stability-indicating assay methods were developed for the determination of tropisetron in a pharmaceutical dosage form in the presence of its degradation products. The proposed techniques are HPLC, TLC, and first-derivative spectrophotometry (1D). Acid degradation was carried out, and the degradation products were separated by TLC and identified by IR, NMR, and MS techniques. The HPLC method was based on determination of tropisetron in the presence of its acid-induced degradation product on an RP Nucleosil C18 column using methanol-water-acetonitrile-trimethylamine (65 + 20 + 15 + 0.2, v/v/v/v) mobile phase and UV detection at 285 nm. The TLC method was based on the separation of tropisetron and its acid-induced degradation products, followed by densitometric measurement of the intact spot at 285 nm. The separation was carried out on silica gel 60 F254 aluminum sheets using methanol-glacial acetic acid (22 + 3, v/v) mobile phase. The 1D method was based on the measurement of first-derivative amplitudes of tropisetron in H2O at the zero-crossing point of its acid-induced degradation product at 271.9 nm. Linearity, accuracy, and precision were found to be acceptable over concentration ranges of 40-240 microg/mL, 1-10 microg/spot, and 6-36 micro/mL for the HPLC, TLC, and 1D methods, respectively. The suggested methods were successfully applied for the determination of the drug in bulk powder, laboratory-prepared mixtures, and a commercial sample.     

An efficient and simultaneous analysis of caffeine and paracetamol in pharmaceutical formulations using TLC with a fluorescence plate reader

A simple, rapid, and efficient method using TLC with a fluorescence plate reader has been described for simultaneous determination of caffeine and paracetamol. Determination was carried out using the fluorescence-quenching action of caffeine and paracetamol on a TLC plate with a fluorescent indicator at lambda ex = 254 nm in the linear ranges of 0.2-1.9 and 0.03-1.5 microg/L, respectively. Separation of caffeine and paracetamol were performed on the TLC plate, and the best results were obtained using the optimized mobile phase n-hexane-ethyl acetate-ethanol (2.5 + 1.5 + 0.4, v/v). Some important parameters, such as solvent type and ratio of the mobile phase, the presence of other components, and instrumental parameters, were studied. Caffeine and paracetamol detection limits were 0.025 and 0.032 microg/L, and RSD values for 0.6 microg/L caffeine and 0.06 microg/L paracetamol (n = 5) were 1.93 and 2.06%, respectively. Using this technique, some pharmaceuticals containing caffeine and paracetamol were analyzed with satisfactory results.     

高效液相色谱法测定强化食品中维生素C的含量

建立了强化食品(饮料、奶粉、含乳饮料、大米、果泥及果冻)中维生素C含量的高效液相色谱检测方法。优化了样品处理方法,在水浴控温和避光条件下处理样品,避免维生素C被氧化。选用Tech Mate C18–ST(250 mm×4.6 mm,5μm)反相色谱柱,以0.05 mol/L磷酸二氢钾缓冲溶液(p H 3)为流动相,流量为1.0m L/min,检测器为光电二极管阵列检测器,检测波长为266 nm。线性范围为0.2~100μg/m L,相关系数为0.999 6,果泥中维生素C的定量限为20 mg/kg,其它为100 mg/kg,加标回收率为82.2%~107%,测定结果的相对标准偏差为1.23%~6.86%(n=8)。该方法简单快速,其灵敏度、准确度和精密度均能满足强化食品中维生素C的检测要求。     

Simultaneous determination of artificial sweeteners, preservatives, caffeine, theobromine and theophylline in food and pharmaceutical preparations by ion chromatography.

A novel ion chromatographic method was proposed for the simultaneous determination of artificial sweeteners (sodium saccharin, aspartame, acesulfame-K), preservatives (benzoic acid, sorbic acid), caffeine, theobromine and theophylline. The separation was performed on an anion-exchange analytical column operated at 40 degrees C within 45 min by an isocratic elution with 5 mM aqueous NaH2PO4 (pH 8.20) solution containing 4% (v/v) acetonitrile as eluent, and the determination by wavelength-switching ultraviolet absorbance detection. The detection limits (signal-to-noise ratio 3:1) for all analytes were below the sub-microg/ml level. Under the experimental conditions, several organic acids, including citric acid, malic acid, tartaric acid and ascorbic acid, did not interfere with the determination. The method has been successfully applied to the analysis of various food and pharmaceutical preparations, and the average recoveries for real samples ranged from 85 to 104%. The levels of all analytes determined by this method were in good agreement with those obtained by the high-performance liquid chromatographic procedure. The results also indicated that ion chromatography would be possibly a beneficial alternative to conventional high-performance liquid chromatography for the separation and determination of these compounds.     

高效液相色谱法同时测定食品接触材料中抗氧化剂和紫外吸收剂的迁移量

采用高效液相色谱技术,建立了食品接触材料中多种抗氧化剂和紫外吸收剂迁移水平的检测方法。该方法测定的23种目标化合物具有较好的线性关系,相关系数（r²）≥ 0.9998,检出限和定量限分别在0.01到0.22 mg/L之间和0.03到0.85 mg/L之间。依据欧盟指令（EU）No. 10/2011,考察了5种食品模拟物30 g/L乙酸、10%（v/v）乙醇、20%（v/v）乙醇、50%（v/v）乙醇和油类模拟物（异辛烷）中抗氧化剂和紫外吸收剂的迁移量。该方法回收率在92.8%~117.7%之间,相对标准偏差在0.95%~9.72%之间。探讨了不同实验条件对抗氧化剂和紫外吸收剂回收率的影响。结果表明,该方法准确、稳定,完全满足欧盟指令（EU）No 10/2011和GB 9685-2008对食品接触材料及制品中抗氧化剂和紫外吸收剂特定迁移量（SML）的限量要求,并利用该方法测定了30批次食品接触材料中抗氧化剂和紫外吸收剂的迁移水平。     

Simultaneous spectrophotometric kinetic determination of four flavor enhancers in foods with the aid of chemometrics

A sensitive kinetic spectrophotometric method was developed for the determination of four flavor enhancers--maltol, ethyl maltol, vanillin, and ethyl vanillin--in food samples. The method was based on the reduction of iron(III) by the four analytes in a sulfuric acid medium (0.012 mol/L), and the subsequent interaction of iron(II) with hexacyanoferrate(III) to form the strongly colored Prussian blue complex, which exhibited an absorption maximum at 800 nm. The optimized method had linear calibrations over the concentration ranges of 0.2-2.8 mg/L for maltol, ethyl maltol, and vanillin, as well as 0.2-1.8 mg/L for ethyl vanillin; the corresponding detection limits were 0.07, 0.07, 0.06, and 0.06 mg/L, respectively. Calibration models were constructed from the original and first-derivative spectral data with the use of partial least-squares (PLS) and principal component regression chemometrics methods. Ultimately, the proposed analytical procedure was successively applied for the determination of the four compounds in commercial food samples with the use of a PLS calibration based on the first-derivative spectral data. The results were comparable with those from a reference HPLC method.     

Simultaneous determination of caffeine and its primary demethylated metabolites in human plasma by high-performance liquid chromatography.

An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its three primary metabolites (theophylline, theobromine and paraxanthine) in human plasma is described. The four substances were separated on a reversed-phase column (5 microns TSK gel ODS-80TM, 150 mm x 4.6 mm I.D.) by use of the mobile phase methanol-0.1 M NaH2PO4 (30:70, v/v) with a flow-rate of 0.8 ml/min. Absorbance was monitored at 274 nm. The detection limit was 5 ng/ml for theobromine and caffeine and 10 ng/ml for paraxanthine and theophylline. The linearity and reproducibility were sufficient for drug monitoring of caffeine and its primary methylxanthines.     

固相萃取-高效液相色谱法同时测定食品塑料包装材料中9种光稳定剂

建立了同时测定食品塑料包装材料中9种紫外光稳定剂含量的高效液相色谱方法。样品用甲醇-乙酸乙酯混合溶剂超声提取,经固相萃取小柱净化后,以ZORBAX SB-C18柱(250 mm×4.6 mm, 5 μm)为分离色谱柱,甲醇和水为流动相,梯度洗脱,以310 nm为检测波长进行定性、定量分析。该方法前处理简单、易操作,9种紫外光稳定剂分离效果良好。9种紫外光稳定剂在0.2～10 mg/L范围内呈良好的线性关系,线性相关系数大于0.999;方法检出限为0.05～0.1 mg/L;实际样品中的加标回收率为70.2%～89.0%,相对标准偏差为0.4%～4.5%。该方法简单、准确,能够满足食品塑料包装材料中紫外光稳定剂的检测要求。     

统计模拟分光光度法同时测定复方茶碱片中的五组分含量

研究了测定复方茶碱片中五组分含量的统计模拟分光光度法。利用逐步回归分析法建立经验数学模型。再运用改良单纯形法进行优化，同时测定复方茶碱片中的五种组分-氨基比林、非那西丁、茶碱、可可碱和咖啡因的含量，其回收率分别为99.5%、100.2%、101.2%、100.2%和99.2%，RSD分别为1.51%、1.03%、4.44%、2.47%和3.12%。t检验结果证明，该方法与HPLC法之间无显著性差异。