Determination of ginsenosides in Panax ginseng roots by liquid chromatography with evaporative light-scattering detection

A liquid chromatographic method was developed and validated for the determination of ginsenosides in Panax ginseng roots by using evaporative light-scattering detection. Eighteen ginsenosides were separated on a reversed-phase C18 column with water-ammonium acetate-acetonitrile as the mobile phase. The method is suitable for the routine determination of ginsenosides in P. ginseng roots and extracts. The validation of the method was comprehensive for efficiency and recovery optimization of the P. ginseng roots extraction, specificity by liquid chromatography/mass spectrometry, linearity, stability, reproducibility, repeatability, intermediate precision, and robustness.     

固相萃取-超高效液相色谱法测定人参中5种原人参二醇型人参皂苷的含量

建立了固相萃取-超高效液相色谱测定人参中5种原人参二醇型人参皂苷的方法。人参药材经粉碎后通过水饱和正丁醇溶液进行超声提取,经过亲水作用固相萃取柱净化后,在ACQUITY UPLC BEH Shield RP18色谱柱(100 mm×2.1 mm, 1.7μm)上分离,利用乙腈/水作为流动相进行梯度洗脱,采用光电二极管阵列检测器检测。结果表明,5种原人参二醇型人参皂苷在5～500μg/mL范围内具有很好的线性关系,相关系数均大于0.999。方法精密度的RSD值在0.95%～2.62%(n=6)之间,22 h内样品稳定性的RSD值在0.90%～2.15%(n=8)之间,日内和日间重复性的RSD值分别为5.35%～6.47%(n=6)和5.56%～6.34%(n=8)。方法的加标回收率在87.16%～101.92%之间,相对标准偏差在1.54%～4.01%(n=6)之间。所建立的方法采用亲水作用色谱模式的固相萃取材料,药材的提取液可直接作为固相萃取的上样溶液进行人参皂苷的富集和净化,并且超高效液相色谱大大缩短了分析时间。该方法简单快速、通量高、重现性好,适用于人参中5种原人参二醇型人参皂苷的...     

Determination of total vitamin C in fruit juices and related products by liquid chromatography: interlaboratory study

A interlaboratory study was conducted to evaluate a liquid chromatographic (LC) procedure for the determination of total vitamin C in foods at levels of 5-60 mg/100 g. Emphasis was placed on fruit juices, although selected foods were also included in the study. Following dissolution of sample in water, endogenous dehydroascorbic acid was converted to ascorbic acid by precolumn reduction with dithiothreitol at neutral pH. Total ascorbate was determined by C18 reversed-phase LC with a phosphate eluent at pH 2.5, incorporating dithiothreitol to maintain vitamin C in the reduced form, and UV detection at 254 nm. Seven types of fruit juices and foods were tested by 19 collaborators in 7 countries. Three duplicate juices and foods met the criteria for Youden pairs and yielded repeatability relative standard deviation of 5.80-14.66%. Reproducibility relative standard deviation ranged from 6.36 to 35.54% (n = 10) with HORRAT values of 0.82-4.04. The LC method is suitable for routine use in fruit products and foods containing > 5 mg/100 g vitamin C and is recommended for further validation by AOAC INTERNATIONAL and International Fruit Juice Union.     

Determination of polyacetylenes and ginsenosides in Panax species using high performance liquid chromatography

A new HPLC method was developed to separate and identify three polyacetylenes (panaxynol, panaxydol and 1,8-heptadecadiene-4,6-diyne-3,10-diol) found in Panax species. The mobile phase was a linear gradient of 2 : 1 : 3 to 2 : 1 : 1 (v/v/v) methanol/acetonitrile/water in 40 min. HPLC analysis was performed at a flow rate of 1.5 ml/min with UV detection at 254 nm. The contents of the polyacetylenes and ginsenosides in Panax ginseng (white ginseng and red ginseng), P. quinquefolium, P. japonicus, and P. noteginseng were determined using these methods. The species containing the highest polyacetylene content (0.080%) was P. quinquefolium cultivated in Nagano, Japan. Meanwhile, the species with the highest ginsenoside content (9.176%) was P. noteginseng cultivated in Yunnan, China.     

超高效液相色谱-飞行时间质谱快速分析人参根中的皂甙化合物

建立了超高效液相色谱-飞行时间质谱快速分析人参根部提取物中的皂甙类化合物的方法。色谱柱为HSS T3超高效液相色谱柱(100 mm×2.1 mm, 1.8 μm);以15 mmol/L甲酸铵水溶液-乙腈为流动相,采用二元梯度洗脱的方式对人参主根的皂甙提取物进行分离。基于待测目标物的多级质谱碎片离子、精确质量等信息,结合9种人参皂甙标准化合物的多级质谱碎片离子质谱图,共鉴定出人参主根提取物中27种皂甙类化合物。在确定的条件下,以9种人参皂甙标样为研究对象,进行了全面的方法学考察,发现它们的线性范围分别为0.33～9.00 mg/L (Rg1), 0.11～9.00 mg/L (Re), 0.02～2.00 mg/L (Rf), 0.07～6.00 mg/L (Rg2), 0.04～3.00 mg/L (Rb1, Rb3), 0.22～6.00 mg/L (Rc), 0.04～9.00 mg/L (Rb2, Rd);在中等加标浓度时,经内标物峰面积校正的9种皂甙标准化合物的峰面积的相对标准偏差(RSD)不高于11.3%;低、中、高3个质量浓度加标水平的回收率范围分别为90%～100%、98%～104%及96%～103%;最低检出限为3.5～18.5 μg/L。该方法具有高分辨、快捷、简便、可靠等特点,并成功地应用于分析同一产地、不同生长时间的人参干燥主根中皂甙的差异。可以预计此方法可进一步应用于各种人参原料和制品中皂甙的快速测定。     

Determination of deoxynivalenol in cereals and cereal products by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic (LC) method for the determination of deoxynivalenol in a variety of cereals and cereal products at proposed European regulatory limits. The test portion was extracted with water. The sample extract was filtered a applied to an immunoaffinity column. After being washed with water, the deoxynivalenol was eluted with acetonitrile or methanol. Deoxynivalenol was quantitated by reversed-phase LC with UV determination. Samples of artificially contaminated wheat-flour, rice flour, oat flour, polenta, and wheat based breakfast cereal, naturally contaminated wheat flour, and blank (very low level) samples of each matrix were sent to 13 collaborators in 7 European countries. Participants were asked to spike test portions of all samples at a range of deoxynivalenol concentrations equivalent to 200-2000 ng/g deoxynivalenol. Average recoveries ranged from 78 to 87%. Based on results for 6 artificially contaminated samples (blind duplicates), the relative standard deviation for repeatability (RSDr) ranged from 3.1 to 14.1%, and the relative standard deviation for reproducibility (RSDR) ranged from 11.5 to 26.3%. The method showed acceptable within-laboratory and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values < 1.3.     

Determination of parthenolide in selected feverfew products by liquid chromatography

The migraine prophylactic herb feverfew (Tanacetum parthenium L.) is marketed in the United States in a variety of forms and compositions. Although its therapeutic efficacy is still uncertain, the sesquiterpene lactone parthenolide is the constituent recommended to be measured for quality control of feverfew preparations. A validated liquid chromatographic method was developed and used to estimate parthenolide in a number of U.S. feverfew market products formulated as capsules, tablets, or crude powder. The method uses a Lichrosphere 5 C18 column, a mobile phase consisting of 50 mM NaH2PO4 in H2O (solvent A), and CH3CN-MeOH (90 + 10, v/v; solvent B). Elution was run at a flow rate of 1.0 mL/min with a linear gradient of 50-15% A in B over 20 min and UV detection at 210 nm. The correlation coefficient for the calibration curve was 0.9999 over the range of 0.00-0.400 mg/mL. Overall recovery of parthenolide was 103.1%.     

Comparative titration of ginsenosides by different techniques in commercial ginseng products and callus cultures

The ginsenoside content of different ginseng species (Panax ginseng, P. quinquefolium, and P. vietnamensis) from different sources (roots from field-grown plants or from in vitro cultures, cells from solid calluses or from liquid cultures, commercial powders, and suspensions) is evaluated by means of a new high-performance thin-layer chromatography (HPTLC) technique combining an automatic TLC sampler and scanner. The results are compared with those obtained through more classical gross spectrometric and high-performance liquid chromatography (HPLC) techniques. HPTLC and HPLC allow the separation and estimation of the different ginsenosides. For this, HPTLC is faster and simpler than HPLC. Both techniques determine less amounts of ginsenosides than spectrophotometry, which displays overestimated values caused by light absorption by contaminating osides. In vitro cultured cells and roots contain the same ginsenosides as those produced by their mother plants, although at quite lower levels. The culture media also accumulates ginsenosides.     

Determination of four active saponins of Panax notoginseng in rat feces by high-performance liquid chromatography

A method is developed for the determination of ginsenoside Rg1, Rb1, Rd, and notoginsenoside R1 of Panax notoginseng (PNS) in rat feces after oral and intravenous administration of total saponins of PNS. The fecal samples are treated with organic extraction and solid-phase extraction prior to high-performance liquid chromatography. The calibration curves for the four saponins are linear in the given concentration ranges. The precision of the method is in the range of 1.0-10.0% (relative standard deviation), and the accuracy is between 80.0% and 110%. The recoveries of this method are all over 75%. This method is successfully applied to the analyses of fecal samples of rats treated with PNS.     

Determination of atropine (hyoscyamine) sulfate in commercial products by liquid chromatography with UV absorbance and fluorescence detection: multilaboratory study

A liquid chromatographic (LC) method with 2 detection systems for determining atropine (hyoscyamine) sulfate in commercial products was tested in a multilaboratory study. Depending on the type of product, sample solutions are prepared in methanol or methanol-water (1 + 1). The standard solution contains about 1.0 mg atropine sulfate/100 mL and is prepared in the same solvent used in sample preparation. LC separations are performed on a 7.5 cm Novapak silica column. The mobile phase is prepared by mixing 970 mL methanol with 30 mL of a 1% aqueous solution of 1-pentanesulfonic acid, sodium salt. Detection is by 2 systems, UV absorbance detection at 220 nm and fluorescence detection with excitation at 255 nm and emission at 285 nm. The injection volume is 100 or 200 microL. The following materials were used for the study: 2 separate samples of tablets labeled to contain 0.4 mg atropine sulfate, 2 separate samples of extended-release tablets labeled to contain 0.375 mg hyoscyamine sulfate, one sample of atropine sulfate injection labeled to contain 2 mg/mL, and one sample of 1% (v/v) atropine sulfate ophthalmic. Eight participants analyzed 2 separate portions of the 6 samples by both detection systems. A ninth participant analyzed the samples in duplicate but only by UV absorbance detection because of the unavailability of a fluorescence detector. The relative standard deviation (RSD) between laboratories ranged from 1.4 to 3.3% for samples of tablets and injections but higher for ophthalmic solutions (5.1-5.2%). A linearity study was conducted in the originating laboratory before the multilaboratory study with 5 solutions ranging in concentration from 0.80 to 1.20 mg atropine sulfate in 100 mL. Average recoveries were 100.0% by UV absorbance detection and 99.9% by fluorescence detection; the RSDs were 1.1 and 1.2%, respectively.     

Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: development, validation, and interlaboratory study

A reversed-phase liquid chromatography method for determination of maduramicin in feedingstuffs and premixtures was developed, validated, and interlaboratory studied. The extraction solvent was methanol. Maduramicin was detected at 520 nm after postcolumn derivatization with vanillin. Recovery was >90%. The repeatability relative standard deviation (RSDr) in feeds (1-5 mg/kg) and premixtures (500 mg/kg) ranged between 2.7 and 7.7%; the within-laboratory reproducibility was between 3.7 and 8.5%. The limit of quantitation was 2 mg/kg. Other feed additives did not interfere in the assay. The method showed ruggedness against small changes in the extraction conditions, eluant composition, and conditions for postcolumn derivatization. The presence of water in the extraction solvent negatively affected the recovery. In the collaborative study, 5 feeds (4 positive at 2.5-9 mg/kg, 1 blank) and 1 premixture (450 mg/kg) were analyzed by 10 laboratories. The RSDr of the feedingstuffs varied between 3.29 and 8.53%. The HORRAT ranged between 1.10 and 1.98. Recoveries were >90%, except for one participant (80%). One laboratory detected small signals in the blank sample, corresponding to 0.7 and 0.8 mg/kg. For the premixture, the RSDr was 3.15% and the HORRAT was 1.80.     

Determination of semicarbazide in fresh egg and whole egg powder by liquid chromatography/tandem mass spectrometry: interlaboratory validation study

An interlaboratory validation study was conducted according to harmonized protocols to evaluate the effectiveness of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of semicarbazide (SEM) in fresh whole egg and in an industrially processed whole egg powder. The sample was extracted with hydrochloric acid and derivatized with 2-nitrobenzaldehyde, with 1,2-[(15)N2(13)C]SEM as the internal standard. The extract was neutralized and purified on a solid-phase extraction cartridge. SEM was determined by reversed-phase LC with detection by MS/MS. Five fresh egg samples, of which 3 were obtained from hens fed nitrofurazone (NFZ), one was spiked with SEM at 50 microg/kg and one was a blank sample, and 5 industrial whole egg powder samples, of which 3 were spiked with fresh whole egg from hens fed NFZ, one was spiked with SEM at 350 microg/kg, and one was a blank sample, were sent to 15 laboratories in 10 different European countries. Results were obtained from 12 participants. Average recoveries of SEM from the fresh egg and the egg powder samples were 105.3 and 121.3%, respectively. The relative standard deviation for repeatability (RSDr) ranged from 2.9 to 9.3%, and the relative standard deviation for reproducibility (RSDR) ranged from 22.5 to 38.1%. The method showed acceptable within- and between-laboratory precision for both matrixes, as evidenced by the HorRat values, at the target levels for the determination of SEM.     

In-depth profiling,characterization, and comparison of the ginsenosides among three different parts (the root,stem leaf,and flower bud) of Panax quinquefolius L. by ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometry

Analytical and Bioanalytical Chemistry - Despite Panax quinquefolius L. serving as a crucial source for food additives, healthcare products, and herbal medicines, unawareness of the metabolome...     

Simultaneous determination of triterpene saponins in ginseng drugs by high-performance liquid chromatography

A HPLC method for the simultaneous determination of 11 triterpene saponins with four-type aglycones (protopanaxadiol, protopanaxatriol, ocotillol and oleanolic acid types) in Ginseng drugs was developed and validated. Using a gradient of acetonitrile and 10 mM K-phosphate buffer (pH 5.80) as the mobile phase and UV detection at 196 nm, more than 18 ginsenosides with different aglycones were separated satisfactorily within 60 min. The detection limits (signal/noise> or =3) were 0.1 microg for ginsenosides Rb1, Rc, Rd, Re and Rg1, chikusetsusaponin III, and notoginsenoside R2, 0.2 microg for gisenoside Ro and chikusetsusaponin IVa, 0.3 microg for chikusetsusaponin IV, and 3 microg for majonoside R2. The calibration curve of each saponin had a correlation coefficient close to 1. Intra- and interday precisions were less than 2.1% (n=5) and 3.3% (n=15), respectively. The recovery rates of extraction were in the range of 96.4-102.7% for all ginsenosides. By adopting this method, the determinations of 11 ginsenosides in three Ginseng drugs derived from Panax ginseng, Panax vietnamensis var. fuscidiscus and Panax japonicus (Japan) were achieved.     

超高效液相色谱法结合化学计量学分析评价4种商品人参的质量

建立了快速、灵敏、准确的超高效液相色谱方法,用来分析4种商品人参(人参、红参、人参叶、人参须)中12种人参皂苷的含量,并用化学计量学方法评价了商品人参的质量。采用ACQUITY UPLC^TM BEH C₁₈色谱柱(50 mm×2.1 mm, 1.7 μm),以乙腈-水为流动相进行梯度洗脱。对所建立的测定12种人参皂苷的UPLC方法进行了线性方程、准确度、重复性、回收率等方法学考察。采用聚类分析和主成分分析的化学计量学方法对4种商品人参进行分析,评价了其质量。结果表明聚类分析和主成分分析2种化学计量学方法非常适合大样本、多成分的中药材质量分析。     

Determination of trans-galactooligosaccharides in selected food products by ion-exchange chromatography: collaborative study

Eleven collaborating laboratories assayed 7 blind duplicate pairs of food and feed products for tans-galactooligosaccharides. The 7 laboratory sample pairs ranged from low (2%) to high levels (15%). Following the proposed method, the test samples were treated with beta-galactosidase and the released galactose was determined by ion-exchange chromatography. Repeatability standard deviation ranged from 2.9 to 11.6%; reproducibility standard deviation ranged from 4.6 to 11.6%.     

Determination of cholecalciferol (vitamin D3) in selected foods by liquid chromatography: NMKL collaborative study

Results are presented from an NMKL (Nordic Committee on Food Analysis) collaborative study of a method for the determination of cholecalciferol (vitamin D3) in foods. The method is based on the addition of an internal standard (vitamin D2), followed by saponification and extraction with n-heptane. The fraction that contains vitamin D2/D3 is separated by preparative normal-phase liquid chromatography (LC), and the analytes are determined by reversed-phase LC with UV detection at 265 nm. The method was tested by 8 participating laboratories. In this study 6 different matrixes were analyzed for cholecalciferol content: milk, liquid infant formula (gruel), cooking oil, margarine, infant formula, and fish oil. The contents varied from 0.4 to 12 microg/100 g. Three matrixes (milk, gruel, and margarine) were fortified with vitamin D3. In the other matrixes, vitamin D3 was added at 3 different levels at the Swedish National Food Administration. The milk was analyzed as a blind duplicate, whereas the other matrixes were analyzed as split-level pairs. The recoveries from the samples with vitamin D3 added varied from 93 to 102%. The repeatability relative standard deviation (RSDr) values for accepted results varied between 2.2% (fish oil) and 7.4% (cooking oil), whereas the reproducibility relative standard deviation (RSD(R)) values varied between 6.8% (margarine) and 24% (cooking oil).     

Determination of total dietary fiber in selected foods containing resistant maltodextrin by a simplified enzymatic-gravimetric method and liquid chromatography: interlaboratory study in China

An interlaboratory study was conducted in China to validate the modified AOAC Official Method 2001.03 for the determination of total dietary fiber (TDF) in foods containing resistant maltodextrin (RMD), which will be adopted as the National Standard Method of China. The kind of buffer solution, the volume of filtrate evaporation, the volume of eluent for desalting and residual solution after evaporation, etc. were modified, which had been proved to have acceptable accuracy and precision in the routine assay. TDF contents in 3 representative foods and 2 kinds of RMD ingredient (i.e., NUTRIOSE 06 and NUTRIOSE 10) were measured using the modified method in 6 eligible laboratories representing commercial, industrial, and governmental laboratories in China. The results of the interlaboratory study indicated that the intralaboratory repeatability, interlaboratory reproducibility, and precision of the modified method are adequate for reliable analysis of TDF in food containing RMD, as well as resistant dextrin. Compared to AOAC Official Method 2001.03, the modified method is time- and cost-saving.     

Determination of chlormequat and mepiquat in foods by liquid chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry: interlaboratory study

Interlaboratory validation studies have been performed on 2 methods for the determination of chlormequat (CLQ) and mepiquat (MPQ). Both methods used identical extraction procedures and stable isotope internal standardization but differed in the use of liquid chromatography/mass spectrometry (LC/MS) or LC/tandem mass spectrometry (LC/MS/MS) for the determination, the amount of internal standard used, and the expected limit of detection. After addition of deuterated internal standards, CLQ and MPQ were extracted with methanol-water and determined by LC//MS or LC/MS/MS with positive electrospray ionization. Eight European laboratories participated in the LC/MS method study, analyzing mushroom, pear, wheat flour, and fruit puree with residues of CLQ in the range 0.040-1.19 mg/kg and of MPQ in the range 0.041-0.39 mg/kg. For CLQ, the Horwitz ratio (HoRat) values for individual test materials/levels were in the range 0.85-1.13 with a mean of 1.00, showing good method performance. For MPQ, the Ho values for mushroom, pear (both levels), and wheat flour were in the range 0.83-0.94, again indicating good method performance. For the determination of MPQ in infant food (fruit puree) at 0.041 mg/kg, the Ho was 1.7 when a value of 0 reported by one participant was excluded. In the LC/MS/MS study, in which 11 laboratories participated, a separate sample set was analyzed with residues of CLQ in the range 0.007-1.03 mg/kg and of MPQ in the range 0.008-0.72 mg/kg. Ho values for CLQ were in the range 0.27-1.36 and for MPQ in the range 0.51-2.10, all corresponding to acceptable method performance.