Molecularly imprinted polymers with synthetic dummy templates for the preparation of capsaicin and dihydrocapsaicin from chili peppers

In this work, dummy molecularly imprinted polymers with high selectivity and affinity to capsaicin and dihydrocapsaicin are designed using N‐vanillylnonanamide as a dummy template. The performance of dummy molecularly imprinted polymers and nonimprinted polymers was evaluated using adsorption isotherms, adsorption kinetics, and selective recognition capacity. Dummy molecularly imprinted polymers were found to exhibit good site accessibility, taking just 20 min to achieve adsorption equilibrium; they were also highly selective toward capsaicin and dihydrocapsaicin. We successfully used dummy molecularly imprinted polymers as a specific sorbent for selectively enriching capsaicin and dihydrocapsaicin from chili pepper samples. In a scaled‐up experiment, the selective recovery of capsaicinoids was calculated to be 77.8% using solid‐phase extraction. To the best of our knowledge, this is the first example of the use of N‐vanillylnonanamide as a dummy template in molecularly imprinted polymers to simultaneously enrich capsaicin and dihydrocapsaicin.     

Analysis of capsaicin and dihydrocapsaicin in peppers and pepper sauces by solid phase microextraction–gas chromatography–mass spectrometry

A simple method for the analysis of capsaicin and dihydrocapsaicin in peppers and pepper sauces by solid phase microextraction–gas chromatography–mass spectrometry has been developed. A novel device was designed for direct extraction solid phase microextraction in order to avoid damage to the fiber. The analysis was performed without derivatization for the gas chromatography–mass spectrometry analysis. Selection fiber, extraction temperature, extraction time and pH, were optimized. The method was linear in the range 0.109–1.323 μg/mL for capsaicin and 0.107–1.713 μg/mL for dihydrocapsaicin with correlation coefficient up to r = 0.9970 for both capsaicinoids. The precision of the method was less than 10%. The method was applied to the analysis of 11 varieties of peppers and four pepper sauces. A broad range of capsaicin (55.0–25 459 μg/g) and dihydrocapsaicin (93–1 130 μg/g) was found in the pepper and pepper sauces samples (4.3–717.3 and 1.0–134.8 μg/g), respectively.     

Scharf,schärfer,Capsaicin!

Capsaicin, the spiciest hot compound known, is a non‐basic alkaloid of chili peppers, belonging to the genus Capsicum. With 16 million units it is on top of the Scoville scale, a measure for the degree of spicy heat within a food. Capsicum, capsaicin and this scale have already been discussed in this journal in 2010. After botanical and culinary aspects, now a method for the separation of the main capsaicinoids, capsaicin and dihydrocapsaicin, is reported which makes use of the complexation of capsaicin by Ag+ ions on an AgNO₃ impregnated silica gel column. All analytical spectra were recorded. They are reproduced either in the main part or in the supporting information and discussed in detail. Calculations of the structure are compared with data of an own x‐ray analysis of capsaicin. The project is a follow up of the recent book “Classics in Spectroscopy” by S. Berger und D. Sicker (Wiley‐VCH 2009).     

Green approach to the extraction of major capsaicinoids from habanero pepper using near-infrared,microwave, ultrasound and Soxhlet methods,a comparative study

A simple method for the extraction of two major capsaicinoids from habanero peppers, using near-infrared irradiation, microwave irradiation or ultrasound as the energy source and ethanol as the solvent, was compared with Soxhlet extraction. The extraction processes were monitored by gas chromatography/mass spectrometry. The new processes offer better overall yields and a higher ratio of capsaicin to dihydrocapsaicin than Soxhlet extraction. The physical effect of the different energy sources on the peppers was determined using scanning electron microscopy. Extraction of capsaicin and dihydrocapsaicin using near-infrared irradiation, which has not been previously reported, was shown to be a simple and efficient alternative extraction procedure.     

The simultaneous determination of capsaicinoids,tocopherols, and carotenoids in pungent pepper powder

From nutritional points of view, carotenoids, capsaicinoids, and tocopherols are valuable constituents in pungent peppers. A rapid and reliable high performance liquid chromatography (HPLC) method for the simultaneous determination of phytonutrients in spice red peppers and chili products was developed and validated. The method included simultaneous detection by fluorescence and diode-array detectors. The major capsaicinoids, two tocopherols and 43 carotenoid components, were simultaneously separated, detected, and identified in the appointed pepper powder (containing Capsicum annuum and Capsicum frutescens) for method validation. The separation was performed on a Nucleosil C18 reverse phase column and optimized gradient elution. Resolution ranged between 0.96 and 1.46 with the highest values corresponding to γ-tocopherol and α-tocopherol. The limits of detection and quantification of target compounds ranged between 18.77 and 148.08 ng mL^?1. Recoveries were between 89.83–100.26 and 79.72–88.86% when standard materials were spiked at low and high amounts, respectively. The most sufficient extraction of the different phytonutrients was achieved by mixture of methanol and acetone, although it was only slightly better than the mixture of methanol and acetonitrile. These results suggest that the developed method could be used for rapid, one-step determination of a wide range of phytonutrients in chili and pepper powders.     

Determination of capsaicin and dihydrocapsaicin in Capsicum fruit samples using high performance liquid chromatography

The aim of the present study was to determine the content of capsaicin and dihydrocapsaicin in Capsicum samples collected from city markets in Riyadh (Saudi Arabia), calculate their pungency in Scoville heat units (SHU) and evaluate the average daily intake of capsaicin for the population of Riyadh. The investigated samples consisted of hot chillies, red chillies, green chillies, green peppers, red peppers and yellow peppers. Extraction of capsaicinoids was done using ethanol as solvent, while high performance liquid chromatography (HPLC) was used for separation, identification and quantitation of the components. The limit of detection (LOD) of the method was 0.09 and 0.10 μg/g for capsaicin and dihydrocapsaicin, respectively, while the limit of quantification (LOQ) was 0.30 and 0.36 μg/g for capsaicin and dihydrocapsaicin, respectively. Hot chillies showed the highest concentration of capsaicin (4249.0 ± 190.3 μg/g) and the highest pungency level (67984.60 SHU), whereas green peppers had the lowest detected concentration (1.0 ± 0.9 μg/g); green peppers, red peppers and yellow peppers were non pungent. The mean consumption of peppers for Riyadh city population was determined to be 15.5 g/person/day while the daily capsaicin intake was 7.584 mg/person/day.     

Determination of capsaicinoids in Capsicum species using ultra performance liquid chromatography‐mass spectrometry

In the present work, a rapid and sensitive ultra performance liquid chromatography‐mass spectrometry method has been proposed for the analysis of capsaicinoids (nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin) present in different Capsicum samples. Extraction of capsaicinoids was carried out by liquid–liquid extraction using ethanol as an extracting solvent, while the chromatographic separation was achieved by reversed phase C₁₈ column with gradient mobile phase (solvent A: acetonitrile and solvent B: water with 0.1% formic acid). Under the optimum experimental conditions, the linear ranges were 0.5–50 μg/g with correlation coefficient (r²) >0.999 for each capsaicinoids and detection limits were 0.15, 0.05, 0.06, 0.2, and 0.1 μg/g for nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin, respectively. Run‐to‐run and day‐to‐day precisions of the method with relative standard deviations <1.5% were achieved for all analyzed capsaicinoids. The robustness of the method was determined by utilizing different injection volumes of the extracts. Furthermore, to validate the system robustness, a run of high number of capsaicinoids present in different varieties of Capsicum samples was performed in this study. All the capsaicinoids were separated in a time of less than 9 min by employing the proposed method.     

Characterization of Red Peppers (Capsicum annuum) by High-performance Liquid Chromatography and Near-infrared Spectroscopy

Dehydration of peppers (Capsicum annuum) is a widely used preservation method. In this study, sun- and hot air-dried red peppers were analyzed for American Spice Trade Association color units, capsaicin, dihydrocapsaicin, organic acids, and free sugars by high-performance liquid chromatography (HPLC), and soluble solids by near-infrared spectroscopy (NIR). In the validation of HPLC protocols, the relative standard deviations were less than 5%, fulfilling the required criteria of Association of Official Analytical Chemists. The concentrations of capsaicin (80.4?mg kg^?1), dihydrocapsaicin (38.0?mg kg^?1), lactic acid (85.2?mg/100?g), glucose (1.521%), and fructose (3.463%) were slightly higher in sun-dried peppers. Linear discriminant analysis showed that NIR spectroscopy is more useful in discriminating sun- and hot air-dried pepper samples.     

Matrix Solid-Phase Dispersion Extraction and UPLC Determination of Sudan Dyes in Chili Powder

A new method involving matrix solid-phase dispersion (MSPD) extraction and UPLC in conjunction with photodiode array detection was developed for the rapid and simple determination of Sudan dyes in chili powder. Separation of Sudan I, Sudan II, Sudan III, and Sudan IV was achieved within 2 min on the 1.7 μm Acquity UPLC BEH C18 column by using gradient elution with a mobile phase consisting of acetonitrile–water at a flow rate of 0.5 mL min^?1. Optimization of MSPD extraction parameters, such as type of solid sorbent and elution solvent were carried out. Optimal conditions selected for MSPD extraction were 0.25 g of sample, 0.5 g of silica gel as solid sorbent, and 7 mL of acetonitrile–methanol (9:1, v/v) as eluting solvent. Limits of detection ranged between 0.25 and 0.30 mg kg^?1 depending on the dye involved. All analytes provided average recoveries from spiked (at 1, 1.5, and 2 mg kg^?1) chili powder samples ranging from 81 to 106%. The method was applied to the analysis of chili powder samples obtained from different countries.     

Simultaneous LC Determination of Quercetin, Kaempferol and Isorhamnetin in Rabbits after Intragastric Administration of an Ethanol Extract from Pollen Typhae

A simple and rapid reversed-phase LC method was developed and validated for simultaneous determination of three flavonoids, quercetin (QU), kaempferol (KA) and isorhamnetin (IS), in rabbit blood plasma. The plasma was deproteinized using 10% trichloroacetic acid and extracted by n-butanol–acetoacetate solvent prior to LC analysis. The analyte was separated on a reversed-phase column with acetonitrile and 0.1% phosphoric acid in water (27:73, v/v) as mobile phase at a flow-rate of 0.8 mL min^?1, and UV detection wavelength at 369 nm. By this developed method, the concentrations of QU, KA and IS were linearly related to their responses in the range of 0.05–2.5 μg mL^?1. The precision and accuracy for QU, KA and IS in plasma were within ±15% except for the limit of quantitation (LOQ), where they were within ±20%. The validated method has been successfully applied in the pharmacokinetic study of QU, KA and IS in rabbits after intragastric administration of an ethanol extract from traditional Chinese medicine Pollen Typhae.     

1H and 13C NMR data on natural and synthetic capsaicinoids

Capsaicinoids are the compounds responsible for the pungency of chili peppers. These substances have attracted the attention of many research groups in recent decades because of their antinociceptive, analgesic, anti‐inflammatory, and anti‐obesity properties, among others. There are nearly 160 capsaicinoids reported in the literature. Approximately 25 of them are natural products, while the rest are synthetic or semi‐synthetic products. A large amount of NMR data for the capsaicinoids is dispersed throughout literature. Therefore, there is a need to organize all this NMR data in a systematic and orderly way. This review summarizes the ¹H and ¹³C NMR data on 159 natural and synthetic capsaicinoids, with a brief discussion of some typical and relevant aspects of these NMR data. Copyright © 2015 John Wiley & Sons, Ltd.     

A Simple and Sensitive LC Method for Quantification of Cefteram in Human Plasma

A simple and sensitive liquid chromatographic method was developed for quantification of cefteram in human plasma. Amoxicillin was used as an internal standard. The present method used protein precipitation for extraction of cefteram from human plasma. Separation was carried out on a reversed-phase C₁₈ column. The column effluent was monitored by UV detection at 262 nm. The mobile phase was a mixture of methanol and water containing 0.3% v/v triethylamine and 0.6% v/v glacial acetic acid (35:65:0.3:0.6 v/v) at a flow rate of 0.30 mL min^?1. The column temperature was 20 °C. This method was linear over the range of 47.5–4,750.0 ng mL^?1 with determination coefficient greater than 0.99. The mean extraction recovery of cefteram and IS was ≥76.82 and ≥76.49%, respectively, and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for a pharmacokinetic study of cefteram in human.     

Simple, Rapid, and Accurate RP-HPLC Method for Determination of Cystine in Human Urine after Derivatization with Dansyl Chloride

An accurate, simple, and sensitive reversed-phase high-performance liquid chromatographic method, with loratadine as internal standard (IS) and UV detection at 286 nm, has been developed for deterination of cystine in human urine. The major innovations of the method include use of acrylonitrile to protect cysteine from oxidization to cystine, separation of cysteine, as the dansyl derivative, from cystine, and use of isocratic elution instead of gradient elution to reduce the time and cost of serial analysis. The mobile phase was 0.05 M sodium acetate–methanol, 35:65 (v/v), adjusted to pH 3.5 with 2.5 M citric acid, at a flow rate of 1.0 mL min^?1. The retention times of cystine and the IS were 16.6 and 19.9 min, respectively. The limit of detection for cystine was 0.3 mg L^?1. Extraction recovery of cystine was >85.6%. Intra-day and inter-day precision (RSD) for cystine were below 4.3 and 8.5%, respectively. There was no chromatographic interference from other α-amino acids present in mammalian proteins, or from other urine components. The calibration plot for the cystine derivative was linear in the range 1–500 mg L^?1 and the correlation coefficient was 0.9992. The method was validated appropriately and successfully used for determination of cystine in human urine.     

Development and Validation of an HPLC Method for Simultaneous Determination of Capsaicinoids and Camphor in Over-the-Counter Medication for Topical Use

A reverse-phase high-performance liquid chromatography method was developed to determine and quantify capsaicin (trans-8-methyl-N-vanillyl-6- nonenamid), dihydrocapsaicin (8-methyl-N-vanillylnonanamide), and camphor (trimethylbicyclo[2.2.1]heptan-2-one). It is applicable in analyses of over-the-counter (OTC) medications for topical use and raw materials such as chili pepper oleoresin. Chromatographic separation was carried out on a C18 column using an isocratic flow of the mobile phase containing acetonitrile and ultrapure water in a ratio of 2:3, with pH adjusted to 3.2 using glacial acetic acid, and a flow rate of 1.5 mL/min. The concentration of the eluting compounds was monitored by a diode-array detector at a wavelength of 281 nm. The method was evaluated for several validation parameters, including selectivity, accuracy (confidence intervals < 0.05%), repeatability, and intermediate precision. The limit of detection (LOD) was determined to be 0.070 µg/mL for capsaicin, 0.211 µg/mL for dihydrocapsaicin, and 0.060 µg/mL for camphor. The limit of quantification (LOQ) was determined to be 0.212 µg/mL for capsaicin, 0.640 µg/mL for dihydrocapsaicin, and 0.320 µg/mL for camphor. Linearity was set in the range of 2.5–200 µg/mL for capsaicin and dihydrocapsaicin and 25–2000 µg/mL for camphor. The suggested analytical method can be used for quality control of formulated pharmaceutical products containing capsaicinoids, camphor, and propolis.     

Improved Sensing of Capsaicin with TiO2 Nanoparticles Modified Epoxy Graphite Electrode

The presented research focuses on the electrochemical determination of capsaicin, a lipophilic alkaloid which originates hotness in chili peppers. An electrochemical sensor based on epoxy‐graphite composite with the modification of titanium dioxide (TiO₂) nanoparticles is developed for the determination of this alkaloid. The measurements were carried out in glycine buffer at pH 2.5 using cyclic voltammetry. Two linear concentration ranges were obtained from 6 to 75 μM (R=0.99) and from 12 to 138 μM, with a detection limit of 5.34 μM and 11.3 μM capsaicin, for 1^st and 2^nd oxidation peak, respectively. The main advantage of developed sensor is its repeatability and robustness against fouling; the relative standard deviation (RSD) value was 2.53 % (n=10). This voltammetric sensing procedure has successfully been applied to quantify capsaicin in various real samples such as hot chili sauce and pharmaceutical preparations.     

LC-ESI-MS-MS Determination of Rat Plasma Protein Binding of Major Flavonoids of Flos Lonicerae Japonicae by Centrifugal Ultrafiltration

A simple, precise, accurate, selective, and sensitive reversed-phase LC–UV method has been developed for simultaneous analysis of diltiazem and non-steroidal anti-inflammatory drugs (NSAIDs) in the bulk drug, tablet dosage forms, and human serum. Chromatographic separation of the drugs was performed at ambient temperature on a C₁₈ stationary phase with 80:20 (v/v) methanol–water, pH 3.1 ± 0.02, as isocratic mobile phase. The mobile phase flow rate was initially 0.5 mL min^?1 then increased to 1 mL min^?1. All the NSAIDs were well separated from each other and from diltiazem. Total run time was 10 min. The assay was successfully applied to pharmaceutical formulations and serum and there was no chromatographic interference from tablet excipients. The method was linear in the range 1.25–50 μg mL^?1 both for diltiazem and the NSAIDs. The suitability of this HPLC method for quantitative analysis of the drugs was proved by validation in accordance with International Conference on Harmonization (ICH) guidelines. The validation results, and results from statistical analysis of the data, demonstrated the method was reliable.     

液-液萃取-液相色谱-串联质谱法测定“地沟油”中辣椒碱类化合物及丁香酚

建立了“地沟油”中辣椒碱类化合物(包括辣椒素、二氢辣椒素、合成辣椒素)及丁香酚的液-液萃取及液相色谱-串联质谱(LC-MS/MS)检测方法。“地沟油”中辣椒碱类化合物及丁香酚用甲醇萃取,采用SUPEL COSIL ABZ+Plus dC18色谱柱(150 mm×4.6 mm, 5 μm)分离,电喷雾离子源在正、负离子模式下电离,多反应监测(MRM)模式扫描。对辣椒素、二氢辣椒素、合成辣椒素及丁香酚的检出限分别为0.02、0.03、0.03和0.6 μg/L,且在一定的质量浓度范围内线性良好。同一操作人员与不同操作人员间测定的精密度低于5%。该方法专属性强、灵敏、准确,可以作为“地沟油”的判定标准之一。     

Dispersive liquid-liquid microextraction for the isolation and HPLC-DAD determination of three major capsaicinoids in Capsicum annuum L.

Dispersive liquid-liquid microextraction (DLLME) was combined with high-performance liquid chromatography-diode-array detector (HPLC-DAD) for the extraction and quantitation of three major capsaicinoids (i.e. capsaicin, dihydrocapsaicin and nordihydrocapsaicin) from pepper (Capsicum annuum L.). Chloroform (extraction solvent, 100 μL), acetonitrile (disperser solvent, 1250 μL) and 30 s extraction time were found optimum. The analytes were back-extracted into 300 μL of 50 mM sodium hydroxide/ methanol, 45/55% (v/v), within 15 s before being injected into the instrument. Enrichment factors ranged from 3.3 to 14.7 and limits of detection from 5.0 to 15.0 µg g^-1. Coefficients of determination (R²) and %RSD were higher than 0.9962 and lower than 7.5%, respectively. The proposed method was efficiently applied for the extraction and quantitation of the three capsaicinoids in six cultivars of Capsicum annuum L. with percentage relative recoveries in the range of 92.0%–108.0%. DLLME was also scaled up for the isolation of the three major capsaicinoids providing purity greater than 98.0% as confirmed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analysis, which significantly reduced the extraction time and organic solvent consumption.     

Ultrasound-assisted extraction of capsaicinoids from peppers

The development of a rapid, reproducible and simple method of extraction of the majority capsaicinoids (nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin) present in hot peppers by the employment of ultrasound-assisted extraction is reported. The study has covered four possible solvents for the extraction (acetonitrile, methanol, ethanol and water), the optimum temperature for extraction (10–60 °C), the extraction time (2–25 min), the quantity of sample (0.2–2 g), and the volume of solvent (15–50 mL). Under the optimum conditions of the method developed, methanol is employed as solvent, at a temperature of 50 °C and an extraction time of 10 min. The repeatability and reproducibility of the method (R.S.D. < 3%) have been determined. The capsaicinoids extracted have been analysed by HPLC with fluorescence detection and using monolithic columns for the chromatographic separation. The method developed has been employed for the quantification of the various capsaicinoids present in different varieties of hot peppers cultivated in Spain.     

Determination of Dutasteride by LC: Validation and Application of the Method

A simple, rapid, and stability-indicating reversed-phase high-performance liquid chromatographic (LC) method for analysis for dutasteride has been successfully developed. Chromatography was performed on a 150 mm × 4.6 mm C₁₈ column with acetonitrile–water 60:40 (v/v) as isocratic mobile phase at 1.0 mL min^?1. Ultraviolet detection of dutasteride was at 210 nm. Its retention time was approximately 10 min and its peak was symmetrical. Response was a linear function of concentration over the range 0.2–1 μg mL^?1 (R ² = 0.997) and the limits of detection and quantitation were was 0.05 and 0.10 μg mL^?1, respectively. The method was validated for linearity, precision, repeatability, sensitivity, and selectivity. Selectivity was validated by subjecting dutasteride stock solution to photolytic, acidic, basic, oxidative, and thermal degradation. The peaks from the degradation products did not interfere with that from dutasteride. The method was used to quantify dutasteride in pharmaceutical preparations.