High-performance Liquid Chromatography Conditions Research Articles

Evaluation of bioactive flavonoids and antioxidant activity in Pericarpium Citri Reticulatae (Citrus reticulata 'Chachi') during storage.

A simple and accurate method using high performance liquid chromatography (HPLC) with dual wavelength detection was developed to simultaneously determine the contents of one flavanone glycoside (hesperidin) and five polymethoxylated flavones (PMFs: sinensetin, 4',5,7,8-tetramethoxyflavone, nobiletin, tangeretin and 5-O-desmethyl nobiletin) in Pericarpium Citri Reticulatae (Citrus reticulata 'Chachi') ('Chachi' PCR). By modifying the mobile phase compositions and detection wavelengths, an optimal HPLC condition was obtained, under which the calibration curves of all six compounds exhibited good linearity (R2>0.99). For all the tested compounds, the relative standard deviation (RSD) was less than 4%, and the accuracy ranged from 97.58 to 103.2%. The developed method was successfully applied to monitor the changes in the contents of six flavonoids in 'Chachi' PCR during storage at 25°C, over a three year period. Color parameters and antioxidant capacity were also determined to evaluate the sample quality. The contents of hesperidin decreased while all the polymethoxylated flavones and antioxidant activities increased throughout the storage period, demonstrating that polymethoxylated flavones could be used as indices for the quality change of Chachi' PCR during storage. The results from this study suggest that the longer storage periods increased the quality of PCR.

Simultaneous determination of multiple ginsenosides in Panax ginseng herbal medicines with one single reference standard

Background:Root of Panax ginseng C. A. Mey (Renseng in Chinese) is a famous Traditional Chinese Medicine. Ginsenosides are the major bioactive components. However, the shortage and high cost of some ginsenoside reference standards make it is difficult for quality control of P. ginseng.Objective:A method, single standard for determination of multicomponents (SSDMC), was developed for the simultaneous determination of nine ginsenosides in P. ginseng (ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, Rd).Materials and Methods:The analytes were separated on Inertsil ODS-3 C18 (250 mm × 4.6 mm, 5 μm) with gradient elution of acetonitrile and water. The flow rate was 1 mL/min and detection wavelength was set at 203 nm. The feasibility and accuracy of SSDMC were checked by the external standard method, and various high-performance liquid chromatographic (HPLC) instruments and chromatographic conditions were investigated to verify its applicability. Using ginsenoside Rg1 as the internal reference substance, the contents of other eight ginsenosides were calculated according to conversion factors (F) by HPLC.Results:The method was validated with linearity (r2 ≥ 0.9990), precision (relative standard deviation [RSD] ≤2.9%), accuracy (97.5%–100.8%, RSD ≤ 1.6%), repeatability, and stability. There was no significant difference between the SSDMC method and the external standard method.Conclusion:New SSDMC method could be considered as an ideal mean to analyze the components for which reference standards are not readily available.SUMMARY A method, single standard for determination of multicomponents (SSDMC), was established by high-performance liquid chromatography for the simultaneous determination of nine ginsenosides in Panax ginseng (ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, Rd)Various chromatographic conditions were investigated to verify applicability of FsThe feasibility and accuracy of SSDMC were checked by the external standard method. Abbreviations used: DRT: Different value of retention time; F: Conversion factor; HPLC: High-performance Liquid Chromatography; LOD: Limit of detection; LOQ: Limit of quantitation; PD: Percent difference; PPD: 20(S)-protopanaxadiol; PPT: 20(S)-protopanaxatriol; RSD: Relative standard deviation; SSDMC: Single Standard for Determination of Multicomponents; TCM: Traditional Chinese Medicine.

Sulfur speciation with high performance liquid chromatography as a tool for El Chichón volcano, crater lake monitoring

The monitoring of sulfur species in crater lakes has proven to be useful for forecasting episodes of volcanic unrest in certain active volcanoes, including Poás, Costa Rica; Kusatsu-Shirane, Japan; and Mt. Ruapehu, New Zealand. In this study, we have improved the current geochemical monitoring of El Chichón volcano through the setting of optimal high-performance liquid chromatography conditions (HPLC) for the analysis of S2−, SO32−, S2O32−, S4O62− and SO42− using a common chromatographic system. The procedure was applied to the analysis of lake samples taken in March, July and October of 2014 and April of 2015. The results were promising, since nearly all species were detected (with the exception of S2O32−) in measurable amounts, including S2− (<0.85–5.05 mg/L), SO32− (<2.77–26.1 mg/L), S4O62− (108.27–303.82 mg/L) and SO42− (489.58–676.26 mg/L). The spatial distribution of these species along the lakeshore showed zones of increased concentrations to the east and southeast of the lake, which provides information on the distribution of faults or cracks that feed hydrothermal fluids to the lake. This method thus provides additional information linked to the volcanic and hydrothermal activity of the volcano.

Method development and validation for dieckol in the standardization of phlorotannin preparations

Phlorotannins are reported to have diverse biological properties. However, no analytical methods for the standardization of phlorotannin preparations have been reported. Herein, we developed and validated an analytical method for the determination of dieckol in phlorotannin extracts (PRT) using high-performance liquid chromatography (HPLC). The optimum HPLC conditions consisted of a Supelco Discovery C18 column stationary phase, a mobile phase (A: 15 % HPLC grade methanol in deionized water, B: methanol), UV detection at 230 nm, and a flow rate of 0.7 mL/min. The optimized chromatographic conditions were validated and exhibited good specificity and linearity (R 2 > 0.9994–1.0000). The recoveries were in the range of 100.9–102.3 %. The method had good intermediate (%RSD 1.2) and intra-day (%RSD 0.4–1.7) assay precisions. This HPLC method had good accuracy and quality in the determination of dieckol in PRT.

Determination of Curcuminoids and Their Degradation Products in Turmeric (Curcuma longa) Rhizome Herbal Products by Non-aqueous Capillary Electrophoresis with Photodiode Array Detection

Non-aqueous capillary electrophoresis (NACE) method was optimised for simultaneous determination of major bioactive curcuminoids (CCD) viz., curcumin (CC), desmethoxycurcumin (DC) and bisdesmethoxycurcumin (BC), and some of the degradation products viz., vanillin (VN), vanillic acid (VA), ferulic acid (FA) and 4-hydroxybenzaldehyde (HB) in turmeric milk and herbal products. Separation was achieved using non-aqueous background electrolyte (NA-BGE), consisting of a mixture of sodium tetraborate, sodium hydroxide, methanol and 1-propanol in a single run by capillary electrophoresis (CE). In the present study, a novel ultrasonication-assisted phase separation method (US-PS) was used for extracting the analytes in turmeric milk and the extract was directly injected into the capillary without any pretreatment. High-performance thin layer chromatographic (HPTLC) method for the separation of the analytes was also evaluated on pre-coated silica gel plate. Two different high-performance liquid chromatographic (HPLC) conditions were reported in the literature for the separation of above-mentioned target compounds. The proposed method NACE is simple, fast, convenient and economical.

Benzo[c]fluorene in Urban Air: HPLC Determination and Mutagenic Contribution Relative to Benzo[a]pyrene.

Benzo[c]fluorene (BcFE) concentrations in benzene/ethanol extracts of airborne particulates were determined by high-performance liquid chromatography (HPLC) with fluorescence detection. HPLC conditions were as follows: columns, two ZORBAX Eclipse PAH (4.6 i.d. × 250 mm, 3.5 μm) and one Inertsil ODS-P (4.6 i.d. × 250 mm, 5 μm) in series; mobile phase, acetonitrile-water (98:2, v/v), 0.3 mL/min; detection wavelengths, excitation 309 nm, emission 354 nm. Particulate-phase BcFE concentrations in the atmosphere varied seasonally (winter > summer). The concentrations were 11000 ± 6100 pg m(-3) (winter) and 40 ± 12 pg m(-3) (summer) in Beijing, China, and 13 ± 5.0 pg m(-3) (winter) and 2.7 ± 0.52 pg m(-3) (summer), in Kanazawa, Japan. In both cities, the particulate-phase BcFE concentration in the atmosphere was lower than that of benzo[a]pyrene (BaP) by a factor of 0.03 - 0.43. However, the mutagenic contribution of particulate-phase BcFE in the atmosphere in winter calculated from the mutagenicity relative potency factor was greater than that of BaP.

How Much Can We Learn from a Single Chromatographic Experiment? A Bayesian Perspective.

In this work, we proposed and investigated a Bayesian inference procedure to find the desired chromatographic conditions based on known analyte properties (lipophilicity, pKa, and polar surface area) using one preliminary experiment. A previously developed nonlinear mixed effect model was used to specify the prior information about a new analyte with known physicochemical properties. Further, the prior (no preliminary data) and posterior predictive distribution (prior + one experiment) were determined sequentially to search towards the desired separation. The following isocratic high-performance reversed-phase liquid chromatographic conditions were sought: (1) retention time of a single analyte within the range of 4-6 min and (2) baseline separation of two analytes with retention times within the range of 4-10 min. The empirical posterior Bayesian distribution of parameters was estimated using the "slice sampling" Markov Chain Monte Carlo (MCMC) algorithm implemented in Matlab. The simulations with artificial analytes and experimental data of ketoprofen and papaverine were used to test the proposed methodology. The simulation experiment showed that for a single and two randomly selected analytes, there is 97% and 74% probability of obtaining a successful chromatogram using none or one preliminary experiment. The desired separation for ketoprofen and papaverine was established based on a single experiment. It was confirmed that the search for a desired separation rarely requires a large number of chromatographic analyses at least for a simple optimization problem. The proposed Bayesian-based optimization scheme is a powerful method of finding a desired chromatographic separation based on a small number of preliminary experiments.

Regulated bioanalysis of conformers - A case study with ASP2151 in dog plasma and urine.

We developed and validated bioanalytical methods for a potent helicase-primase inhibitor ASP2151 that has two conformers. These conformers elute as unseparated broad peaks under ordinary high-performance liquid chromatographic conditions, indicating discernable differences in hydrophobicity. We observed that column temperature and mobile phase pH have no effect on these peaks and that conformers form a single symmetrical peak when tetrahydrofuran is added to the mobile phase. In addition, we needed to develop semi-automated methods where inter-conversion of the conformers is unlikely to cause sample-to-sample extraction variability. Briefly, following the addition of deuterium-labeled ASP2151 as an internal standard (IS), dog plasma samples or acetonitrile-added urine samples were filtrated. The filtrates were then injected into a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and trapped onto an extraction column. Extracts were back-flushed onto an analytical C18 column (4.6×50mm, 3μm) with a mobile phase consisting of methanol, tetrahydrofuran, and 20mmol/L ammonium acetate (45:5:50, v/v/v). The eluent was monitored in the negative atmospheric pressure chemical ionization mode. The calibration curve was linear over a range of 5-1000ng/mL for plasma and 0.5-100μg/mL for urine. Validation data met the acceptance criteria in accordance with regulatory guidance and demonstrated that these methods were selective, accurate, and reproducible. In addition, the present methods were successfully applied to a pharmacokinetic study in dogs.

Synthesis and evaluation of dummy molecularly imprinted microspheres for the specific solid-phase extraction of six anthraquinones from slimming tea.

Dummy molecularly imprinted microspheres with danthron as template were synthesized and their performance was evaluated. Accelerated solvent extraction can rapidly and effectively remove template molecules from the microspheres. The microspheres were applied as a specific sorbent for solid-phase extraction of six anthraquinones from slimming tea, showing excellent affinity and high selectivity to danthron and the target analytes. The molecular recognition mechanisms were discussed by the experimental validation with IR spectroscopy. The sample was treated using accelerated solvent extraction followed by dummy molecularly imprinted microspheres solid-phase extraction. Under the optimized ultra high performance liquid chromatographic conditions, the six target analytes can be baseline separated in 8 min, and good linearity was obtained in a range of 0.1-40 μg/mL with the correlation coefficient (r(2)) of ≥0.9998. The method limit of quantification was in a range of 1-2 mg/kg, it can ensure analysis of anthraquinones at mg/kg level. The intra- and interday precision (RSD, n = 6) for the analysis of the six analytes in a slimming tea was less than 4.5 and 5.4%, respectively. The developed method can be applied for the selective extraction, effective separation, and rapid determination of six anthraquinones in slimming tea.

Determination of berberine in Phellodendron amurense from different sites of Changbai Mountain

Phellodendron amurense has been used for many years as a medical plant in traditional Chinese medicine and has shown great prospect in recent clinical trials for future applications. Berberine is an essential active compound contained in P. amurense. Our objective in this study was to quantify the content of berberine in P. amurense from sites at different elevations on Changbai Mountain. We collected samples of P. amurense from five different elevations on Changbai Mountain. Berberine in samples was extracted by ultrahigh pressure extraction (UPE). And the quantity was measured by high performance liquid chromatography (HPLC). First, the optimal HPLC conditions for berberine were identified with satisfactory precision (relative standard deviation, R SD < 5.6 %), good accuracy (relative error, R E < 3.6 %) and good linear relation (R 2 = 0.9998) in the range of 6.576–328.8 mg·L−1. Second, the combination of UPE and HPLC methods in quantitative analysis of berberine showed high repeatability (R SD = 3.28 %), reproducibility (R SD = 4.72 %), stability (R SD < 1.27 %) and good recovery (99.54 %) for real plant materials. Samples from Heilongjiang Province at the lowest elevation contained the highest amount of berberine. Similarly, the lowest amount of berberine was recorded in samples from Changbai Forest Bureau of Jilin Province collected at the highest elevation in this paper. The proposed UPE–HPLC method is simple, reliable and low-cost for quantitative analysis of berberine. Content of berberine in P. amurense varied significantly by site on Changbai Mountain.

Solid-phase extraction based on mesoporous Fe 3 O 4 @mSiO 2 @Cu 2+ magnetic nanoparticles coupled with high performance liquid chromatography for the determination of microcystins in water samples

Mesoporous magnetic nanoparticles of Fe3O4 @ mSiO2 @ Cu2+ were synthesized by two methods of hydrothermal synthesis and room temperature synthesis. The synthesized mesoporous Fe3O4 @ mSiO2 @ Cu2+ nanoparticles presented uniform sizes, good magnetic properties and specific selectivity. In this work, a novel magnetic solid-phase extraction (MSPE) method based on mesoporous Fe3O4 @ mSiO2 @ Cu2+ nanoparticles (NPs) coupled with high performance liquid chromatography (HPLC) was developed for the simultaneous extraction of trace amount of microcystin-LR (MC-LR) in water samples. The MSPE extraction conditions and HPLC conditions were optimized. The results showed that good linear relationship (r = 0.999 4) was obtained between peak area and mass concentration of MC-LR in the range of 0.1-15 µg/L. The limit of detection was 0.025 µg/L, and the limit of quantification was 0.082 µg/L. Furthermore, this method was used for the analysis of algal toxins in water samples with the recovery of 78%. The results indicated that Fe3O4 @ mSiO2 @ Cu2+ magnetic nanoparticles were good pretreatment alternatives for the concentration of microcystin-LR in water samples.

In Vitro Ceftriaxone Stability at New-borns’ Rectal PH Assessed by UV and HPLC Methods

This study showed the comparison of UV spectroscopy and High Performance Liquid Chromatography (HPLC) for ceftriaxone stability. UV spectroscopy using wavelength ratio between 241 and 271 nm absorbance values can be used successfully as a screening technique in ceftriaxone stability investigations. Ion paring reversed phase – High Performance Liquid Chromatography provided more precise stability characterization. The HPLC conditions developed were in isocratic mode using an YMC ODS H80, 150 x 4.6 mm, 4 μm with a mobile phase composed by 40% of methanol and 60% of phosphate buffer (10 mM; pH 7.5) where tetrabutylammonium bromide was solubilized at 18 mM. Detection was performed with a diode array detector from 200 to 400 nm. Sample injection volume was at 5 μL. Methanol was selected because better symmetry of ceftriaxone peak than acetonitrile was obtained. Both methods were validated. The calibration curve and stability study was performed over a concentration range of 7.5 to 16.5 mg.L-1. 100% corresponded to the concentration of 15 mg.L-1. Intermediate precision was tested on 6 independent samples at concentrations corresponding to 100% (15 mg.L-1) on 6 consecutive days. These values were within the acceptance criteria of 2% and showed that both methods were precise. Accuracy of the method was evaluated analyzing three independent samples at concentrations corresponding to 100%. Recovery percentage calculated between the known concentration and the calculated concentration of ceftriaxone showed that the methods were accurate. Thus both methods were linear. The stability study was performed at 40°C as infants with sepsis are generally febrile. Over the rectal pH range recorded in sick infants, the stability of ceftriaxone was maximal at pH 7.5. Over 6 hours in a pH range of 6.5 to 8.5 less than 10% of ceftriaxone is degraded. However at pH 5.5, degradation occurred more rapidly and loss of drug was significant.

HPLC in simultaneous determination of seven main flavonoids in Epimedium of different places of origin

Objective To determine the contents of seven main flavonoids in Epimediumof different places of origin by high performance liquid chromatography(HPLC).Methods The HPLC condition was as follows:SHISEIDO MG-C18column(3.0mm×100 mm,3.0μm);the mobile phase consisted of acetonitrile and water containing 0.1% formic acid was used as gradient elute;the gradient of acetonitrile was 25%(0-10min),25%-40%(10-12min),40%-45%(12-22min),45%-75%(22-25min)and 75%(25-30 min);flow rate was 0.6 mL/min;UV detection wavelength was set at 270 nm;column temperature was 25℃;and injection volume was 5μL.The sample was ultrasonically extracted with 70% ethanol solution.Results The seven flavonoids,including epimedin A,epimedin B,epimedin C,icariin,icariside Ⅰ,icariside Ⅱ and anhydroicaritin were separated at baseline within 30 min.The standard curves had good linearity(r=0.999 9),with the RSD%of intraday and interday precision being both less than 2.0%,the recovery being 98%-102%,and the RSD% of stability and repeatability also being less than 2.0%.The results showed that the method could meet the requirements of method validation.The contents of seven flavonoids in reference crude meterial and Herba Epimedii from Liaoning,Gansu and Hubei province were determined.Conclusion The present method is rapid and simple,and can be used for quality control of Epimedium,which may lay a foundation for studying the pharmacokinetics and tissue distribution of the flavonoids of Epimedium.

Multi-component analysis in sun-dried and sulfur-fumigated Angelicae Sinensis Radix by single marker quantitation and chemometric discrimination.

Background:A new method has been developed for the simultaneous determination of ferulic acid, senkyunolide A, and Z-ligustilide in Angelicae Sinensis Radix before and after sulfur-fumigation using quantitative analysis of multi-components by a single marker (QAMS).Materials and Methods:The feasibility and accuracy of QAMS were checked by the external standard method, and various high-performance liquid chromatographic instruments and chromatographic conditions were investigated to verify its applicability. Using ferulic acid as the internal reference substance, and the contents of senkyunolide A and Z-ligustilide were calculated according to relative correction factors by high-performance liquid chromatography. Meanwhile, the influence of sulfur-fumigation on these chemical components in Angelicae Sinensis Radix were evaluated and discriminated by chromatographic fingerprint and chemometrics.Results:There was no significant difference observed between the QAMS method and the external standard method. Furthermore, sulfur-fumigation reduced the contents of ferulic acid, senkyunolide A, and Z-ligustilide in Angelicae Sinensis Radix by some degree, and the sun-drying and sulfur-fumigation processing could be easily discriminated by chromatographic fingerprint and chemometrics.Conclusion:QAMS is a convenient and accurate approach to analyzing multi-component when reference substances are unavailable, simultaneously, chemometrics is an effective way to discriminate sun-dried and sulfur-fumigated Angelicae Sinensis Radix.

Quantification of Inulin Content in Selected Accessions of Jerusalem Artichoke (Helianthus tuberosus L.)

Jerusalem artichoke is an excellent source of inulin. Inulin has valuable nutritional and functional attributes, and therefore it is needed to know inulin content in different accessions of Jerusalem artichoke tubers. We used rapid high-performance liquid chromatography (HPLC) method following water extraction to determine inulin content in Jerusalem artichoke tubers. HPLC conditions included Rezex RCM-monosaccharide Ca2+ column, deionized water as mobile phase and light scattering detection. It was found that inulin content of Jerusalem artichoke tubers ranged from 8.16 to 13.46% of fresh weight. The maximum value of inulin content in 12 accessions of Jerusalem artichoke was detected in TUB CG 32.

Development and validation of an ultrasound-assisted surfactant-enhanced emulsification microextraction method for liquid chromatographic determination of carbamate residues in fruit juices

A microextraction procedure based on ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME) was optimized and validated for preconcentration of carbamate pesticides prior to fast determination using high-performance liquid chromatography (HPLC) with monolithic column. The nonionic surfactant (Tween 20) was used as an emulsifier, and chloroform was selected as an extraction solvent. The experimental parameters affecting the extraction efficiency, such as the kind, concentration, and volume of surfactant, the kind of extraction solvent and its volume, salt addition, ultrasonic extraction time, and centrifugation time, were optimized. Under the selected UASEME and HPLC conditions, separation of five carbamate pesticides (carbofuran, carbaryl, isoprocarb, methiocarb, and promecarb) was achieved within 10 min. The linearity of the method was obtained in the wide range of 2–5000 μg L−1, with correlation coefficients of 0.9931–0.9970. The limits of detection were in the range of 0.4–7.0 μg L−1. The relative standard deviations (n = 5) were less than 0.59% and 4.86% for retention time and peak area, respectively. The enrichment factors of the developed UASEME method were in the range of 33–166. The fruit juice samples (at fortified levels of 0.25, 0.50, and 1.00 mg L−1) were successfully analyzed, and the relative recoveries were obtained in the range of 81–115%.

Purification and Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry of Aroma Compounds from Green Tea Products and Comparison to Bulk Analysis

A method for carbon isotope ratio (δ(13)C) analysis was developed for compound-specific isotope analysis of tea volatiles, and the values were compared with the δ(13)C value from bulk isotope analyses. The δ(13)C value of 2-phenylethanol liberated via enzymatic hydrolysis of the 2-phenylethyl β-primeveroside standard was examined first. Isotope fractionations for 2-phenylethyl β-primeveroside from preparative high-performance liquid chromatography (HPLC) were also analyzed. The enzymatic treatment and the preparative HPLC process did not cause carbon isotope fractionations, substantiating the strategies available for δ(13)C analysis of volatile compounds. On the basis of the gas chromatography-combustion-isotope ratio mass spectrometry data from 2-phenylethanol, it was possible to derive the conditions for enzyme treatment and preparative HPLC of the glycoconjugates of 2-phenylethanol, (Z)-3-hexenol, and benzyl alcohol isolated from green tea leaves. Larger variations in δ(13)C were found for individual volatile compounds compared with bulk analytical data from the leaves, indicating the potential to utilize this strategy in assigning the geographical origin of green tea.

Determination of the trace residues of four organochlorine pesticides in agricultural products by high performance liquid chromatography with modified multi-walled carbon nanotubes as solid phase extraction adsorbent

A novel method for the simultaneous determination of the trace residues of four organochlorine pesticides such as p,p'-DDD, p,p'-DDT, o,p'-DDT and p,p'-DDE in agricultural products by multi-walled carbon nanotubes (MWNTs) modified by acid oxidation on the surface as solid phase extraction (SPE) adsorbent coupled with high performance liquid chromatography (HPLC) was developed. The effects of the surface acid oxidation, SPE operations and HPLC conditions on the determination of the four pesticide residues were investigated. Under the optimized experimental conditions, the novel method provided wide linear ranges for the pesticides with correlation coefficients of 0. 997 8 - 0. 999 5, the detection limit was 0.050 mg/L for each pesticides. The recoveries from the samples spiked with the pesticide standards at three concentration levels of 0.10, 2.0 and 50 mg/L were in the range of 78% - 104% with the relative standard deviations (RSDs) of 2.7% -7.6%. This study indicated the MWNTs SPE was an efficient clean-up method to agricultural products (included dried orange peel, ginseng, cabbage and tea). The proposed method showed the advantages of accuracy and sensitivity, and can meet the requirements for the determination of low residue pesticide in agricultural products. The study provides a useful method for the analysis of trace substance of agricultural products.

Recent Developments in HPLC Analysis of β-Blockers in Biological Samples

β-Adrenergic blockers represent a very important class of drugs that are used worldwide for treating various cardiac diseases. The present article describes the state-of-the art of analyses of β-adrenergic blockers using high-performance liquid chromatography (HPLC). Sample preparation techniques such as liquid-liquid extraction, solid-phase extraction and solid-phase microextraction have been discussed, which are essential prior to HPLC analysis. Additionally, applications of liquid chromatography coupled with tandem mass spectrometry are included. HPLC methods have been reported to include 0.6-26 min as the run times and 0.01 ng/mL to 25 µg/mL as detection limits. The most commonly used columns were C18 with various buffers as the mobile phases, along with various organic modifiers. The optimization of HPLC conditions has been discussed. It has been observed that the reported methods are quite satisfactory for the analyses of β-adrenergic blockers in biological samples. Future perspectives in the hyphenation of solid-phase microextraction-nano-liquid chromatography-tandem mass spectrometry have also been highlighted to achieve detections at nanogram and picogram levels. The present article is very useful for academicians, scientists, drug and pharmaceutical personnel and government regulatory authorities.

Correlation of Normal Phase TLC to HPLC Results in the Isolation of Chlorophyll Degradation Components Extracted from Algae and Extra Virgin Olive Oil

Isolation of high purity examples of naturally occurring compounds is a challenging task for many laboratories accustomed to an analytical chromatography workflow. It may be unavoidable, however, when one discovers that many obscure components are not commercially available in sufficient purity for use as qualitative or quantitative standards in mainstream analytical use. Further, when the components lending observable biological activity in a crude extract have not been identified, the chromatographer depends not only on achieving purification of potentially hundreds of compounds but also on knowing that all of the components in the extract are being successfully recovered from the purification column, and in the sample chemical form as were present in the crude mixture. Thin Layer Chromatography (TLC) can be a useful tool for screening crude extracts and predicting column elution. It can be more effective than High Performance Liquid Chromatography (HPLC), and it is a relatively easy to use tool in the hands of chemists who may not have formal training or experience with HPLC. Translation of the selectivity, retention and resolution observed with TLC methods to an HPLC preparative separation can be challenging. In the presented work, we used extracts from algae and bulk extra virgin olive oil to isolate chlorophyll and degradation components in the pheophytin, pheophorbide and pyropheophytin structural groups. We elaborate the key elements of TLC that allow recovery to be assessed, and how a straightforward conversion to usable HPLC conditions can be achieved. The combined use of UV/VIS Diode Array detection (DAD), Evaporative Light Scattering detection (ELSD) and single quadrupole MS allowed for an effective characterization of structure and purity for the isolated target compounds.