High-performance Liquid Chromatography Peak Research Articles

Analysis of Glucosinolates and Phenolic Content in Sprouts of 7 Brassica rapa Subspecies

Objective: Brassica rapa is an important source of phytochemicals essential for plant defense and human health. To better understand their metabolic characteristics, this study aimed to investigate the glucosinolates and phenolic content from sprouts of 7 B. rapa subspecies. Methods: Glucosinolates and phenolic compounds were extracted using previously reported procedures, with slight modifications. Glucosinolates were identified based on their high-performance liquid chromatography peak area ratios and quantified based on the peak areas, retention time, and response factor and phenolic compounds were identified based on retention times and spiking tests and quantified concerning a corresponding calibration curve. Results: Statistical analysis showed that Choy sum (31.82 ± 2.3 mg/g DW) and Bok choy (31.73 ± 1.49 mg/g DW) presented the highest total glucosinolates content, and the lowest content was observed in Turnip rape (21.75 ± 1.67 mg/g DW). Between the contents of individual glucosinolates, progoitrin presented the highest content in Bok choy (17.84 ± 1.24 mg/g DW). The highest concentration of total phenolic compounds was found in the sprouts of Chinese cabbage (2.7 ± 0.05 mg/g DW), whereas the lowest concentration was found in Choy sum sprouts (2.17 ± 0.05 mg/g DW). The highest individual phenolic content was observed for benzoic acid (0.96 ± 0.03 mg/g DW) and rutin (0.85 ± 0.03 mg/g DW), which were highest in mizuna and Chinese cabbage, respectively. Conclusion: Subspecies may influence the levels of glucosinolates and polyphenols in vegetables. The results of our study showed the distinctive metabolic signatures embedded in the 7 B. rapa subspecies.

The impact of non-environmental factors on the chemical variation of Radix Scrophulariae

Radix Scrophulariae is a commonly used Chinese herb derived from the dried root of Scrophularia ningpoesis Hemsl. (S. ningpoensis). It is difficult to accurately estimate the dosage of Chinese medicinal materials used in the prescription because of the chemical variation caused by various factors. To analyze the non-environmental factors affecting the chemical variation of Radix Scrophulariae, we planted nine different cultivated varieties of S. ningpoensis in the same plantation. Based on sequence-related amplified polymorphism (SRAP), simple sequence repeats (SSR) markers and high-performance liquid chromatography (HPLC) analysis, we found that the materials from the cultivated varieties could be divided into two groups, the Zhejiang group, and the southwest China group. The genetic distance based on molecular data between the two groups was above 0.3882, and the Euclidean distance based on chemical data between the two groups was above 5.312. The correlation analysis between the genetic distance matrix based on SRAP and the Euclidean distance matrix based on 18 HPLC peaks of the whole underground part revealed that the genetic differentiation and chemical variation were positively related, r = 0.7196 (p < 0.05). The genetic background, different part of the roots and the different development of the roots are the three non-environmental factors causing the chemical variation. The coefficient of variation (C.V) of chemical composition of Radix Scrophulariae with different genetic background reached to 93.62 %, the C.V of the chemical composition of Radix Scrophulariae derived from the same variety reached to 64.21 %, the C.V of the chemical composition of Radix Scrophulariae derived from the middle part of the roots of S. ningpoensis from the same variety reached to 45.55 %. The C.V of chemical composition of Radix Scrophulairae produced in the same plantation could be controlled to 38.43 % by using the same variety of roots with the approximate mass derived from the middle part of the roots under 20 g. Our findings provided insights to decrease the chemical variation of Chinese medicinal materials by controlling non-environmental factors.

Nuclear magnetic resonance-based solvent system selection for counter-current chromatography separation of compounds present in the same high-performance liquid chromatography peak: Flavonoids in barley seedlings as an example.

Partition coefficient is a key parameter for counter-current chromatography separation. High-performance liquid chromatography (HPLC) is the most commonly used tool for the screening of partition coefficients. However, HPLC technology is not applicable to the compounds present in the same chromatographic peak. Nuclear magnetic resonance (NMR) technology could easily distinguish compounds according to their characteristic absorption even if they exist in the same HPLC peak. In this study, two flavonoids present in the same HPLC peak were successfully purified by counter-current chromatography with a solvent system screened by NMR to show the great potential of NMR technology in the screening of the partition coefficient of co-efflux compounds. Through NMR screening, an optimized ethyl acetate/n-buthanol/water (7:3:10, v/v/v) system was applied in this study. As a result, two flavonoids, including 4.8mg of 3'-methoxyl-6'''-O-feruloylsaponarin and 9.8mg of 6'''-O-feruloylsaponarin were separated from 15mg of the mixture. There is only one methoxy group difference between the two flavonoids. This study provides a new strategy for the screening of counter-current chromatography solvent systems and broadens the application scope of counter-current chromatography.

Surface-Enhanced Raman Analysis of Uric Acid and Hypoxanthine Analysis in Fractionated Bodily Fluids.

In recent years, the disease burden of hyperuricemia has been increasing, especially in high-income countries and the economically developing world with a Western lifestyle. Abnormal levels of uric acid and hypoxanthine are associated with many diseases, and therefore, to demonstrate improved methods of uric acid and hypoxanthine detection, three different bodily fluids were analysed using surface-enhanced Raman spectroscopy (SERS) and high-performance liquid chromatography (HPLC). Gold nanostar suspensions were mixed with series dilutions of uric acid and hypoxanthine, 3 kDa centrifugally filtered human blood serum, urine and saliva. The results show that gold nanostars enable the quantitative detection of the concentration of uric acid and hypoxanthine in the range 5-50 μg/mL and 50-250 ng/mL, respectively. The peak areas of HPLC and maximum peak intensity of SERS have strongly correlated, notably with the peaks of uric acid and hypoxanthine at 1000 and 640 cm-1, respectively. The r2 is 0.975 and 0.959 for uric acid and hypoxanthine, respectively. Each of the three body fluids has a number of spectral features in common with uric acid and hypoxanthine. The large overlap of the spectral bands of the SERS of uric acid against three body fluids at spectra peaks were at 442, 712, 802, 1000, 1086, 1206, 1343, 1436 and 1560 cm-1. The features at 560, 640, 803, 1206, 1290 and 1620 cm-1 from hypoxanthine were common to serum, saliva and urine. There is no statistical difference between HPLC and SERS for determination of the concentration of uric acid and hypoxanthine (p > 0.05). For clinical applications, 3 kDa centrifugal filtration followed by SERS can be used for uric acid and hypoxanthine screening is, which can be used to reveal the subtle abnormalities enhancing the great potential of vibrational spectroscopy as an analytical tool. Our work supports the hypnosis that it is possible to obtain the specific concentration of uric acid and hypoxanthine by comparing the SER signals of serum, saliva and urine. In the future, the analysis of other biofluids can be employed to detect biomarkers for the diagnosis of systemic pathologies.

Continuous, Nondestructive Detection of Microorganism Growth at Buried Interfaces with Vascularized Polymers.

Evaluating surface bacterial growth at buried interfaces can be problematic due to the difficulties associated with obtaining samples. In this work, we present a new method to detect signals from microorganisms at buried interfaces that is nondestructive and can be conducted continuously. Inspired by vascular systems in nature that permit chemical communication between the surface and underlying tissues of an organism, we created a system in which an inert carrier fluid could be introduced into an empty vascular network embedded in a polymer matrix. When a microorganism layer was grown on top, small molecules produced by the growth process would diffuse down into the carrier fluid, which could then be collected and analyzed. We used this system to nondestructively detect signals from a surface layer of Escherichia coli using conductivity, ultraviolet-visible (UV-vis) absorbance spectroscopy, and high-performance liquid chromatography (HPLC) for organic acids, methods that ranged in sensitivity, time-to-result, and cost. Carrier fluid from sample vascularized polymers with surface bacterial growth recorded significantly higher values in both conductivity and absorbance at 350 nm compared to controls with no bacteria after 24 h. HPLC analysis showed three clear peaks that varied between the samples with bacteria and the controls without. Tests tracking the change in signals over 48 h showed clear trends that matched the bacterial growth curves, demonstrating the system's ability to monitor changes over time. A 2D finite element model of the system closely matched the experimental results, confirming the predictability of the system. Finally, tests using clinically relevant Staphylococcus aureus and Pseudomonas aeruginosa yielded differences in conductivity, absorbance, and HPLC peak areas unique to each species. This work lays the foundation for the use of vascularized polymers as an adaptive system for the continuous, nondestructive detection of surface microorganisms at buried interfaces in both industry and medicine.

Quantitative determination of major alkaloids in areca nut products by high-performance liquid chromatography coupled with ion mobility spectrometry.

Areca nut is a popular and addictive food as well as a traditional herbal medicine in many countries. Areca nut contains alkaloids including arecoline, guvacine, and arecaidine, which are the major bioactive compounds in areca products. Areca alkaloids can be carcinogenic, and thus sensitive and specific analytical methods are urgently desired for the identification and quantification of these compounds. High-performance liquid chromatography-based methods are often preferred, but areca alkaloids do not have chromophores, and detection using a traditional UV detector can be difficult. The complexity of areca sample extracts can also lead to the co-elution of peaks leading to poor quantitative performance. We report here high-performance liquid chromatography coupled with an ion mobility spectrometer for sensitive determination of areca alkaloids in various products including areca nut, areca nut products, and herbal oral liquid. An X-Bridge reversed-phase C18 column was used in the experiment and was combined with high-performance liquid chromatography coupled to an ion mobility spectrometer system. A custom-made adjustable post-column splitter acted as an interface between the high-performance liquid chromatography and the ion mobility spectrometer; it also acted as the electrospray ionization source. The mobile phase was methanol and 0.5% ammonium hydroxide. The results demonstrate that the splitter can afford a wide range of split ratios that match the ion mobility spectrometer ionization source while keeping the separation efficiency of high-performance liquid chromatography. Three major alkaloid compounds were then accurately determined using the resulting method without dativization steps. Many coeluted high-performance liquid chromatography peaks are effectively separated in the ion mobility spectrometer dimension, which in turn improved the quantification accuracy.

An Integration of Statistical and Anthropometric Measurement Approach Towards Improving Ergonomic Design for Production Workbench

Capillary zone electrophoresis (CZE) is a method of haemoglobin (Hb) analysis for the screening of haemoglobinopathies or variant haemoglobins. In our centre, few samples sent for Hb analysis showed Hb peak at zone 10 requiring further evaluation. This pattern was observed in patients with history of diabetes mellitus (DM); hence it was postulated the peak observed is glycated Hb (HbA1c). The objective of this study was to determine whether the expression of Hb peak at zone 10 among blood samples from DM patients correlate with HbA1c values measured using high performance liquid chromatography (HPLC). Methods: This was a cross-sectional study involving blood samples from DM patients. Samples with HbA1c values ≥ 6.5% by HPLC were selected and subsequently analysed by CZE method. Presence of Hb peak in zone 10 were correlated and analysed with the HbA1c levels measured by HPLC method. Results: A total of 131 samples were analysed. Hb peak was detected at zone 10 in 50/131(38.2%). Out of 50 samples, 47 (94%) were from patients with HbA1c level &gt; 10%. Correlation study showed it was 18 times more likely for Hb peak to appear in zone 10 in patients with HbA1c &gt; 10%. Cut off point for HbA1c to appear in CZE is 10.5% with AUC of 0.965. Conclusion: Hb peak detected at zone 10 of CZE was most likely to be HbA1c. However, it is recommended that for every primary method of Hb analysis used be confirmed by secondary method. Therefore, both zone 10 in CZE and P2 peak in HPLC must be correlated together to achieve final diagnosis

A high-performance liquid chromatography-evaporative light scattering detection-based quantitative analytical method for polyvinyl alcohol in food supplements with pyrolysis-gas chromatography/mass spectrometry-based verification.

Polyvinyl alcohol (PVOH) is extensively used in agricultural, pharmaceutical, textile, and food industries. A new high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was developed for the quantitative analysis of PVOH products. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) was employed for method verification. The HPLC-ELSD method exhibited acceptable linearity (R2 > 0.99), with limits of detection and quantitation of 19.43 and 58.88μg/mL, respectively. Accuracies of 91.16-102.30% were estimated based on recoveries of the intra- and inter-day tests of PVOH. Repeatability and intermediate precision (%RSD) of 1.23-4.45 and 2.18-6.95, respectively, were obtained. The presence of PVOH in the HPLC peaks was further verified using typical indicators of PVOH in Py-GC/MS analysis, such as acetaldehyde, 2,5-dihydrofuran, benzaldehyde, and crotonaldehyde. This novel HPLC method with Py-GC/MS-based verification can be successfully applied for analyzing PVOH in dietary supplement tablets.

Eliminating protein oxidation artifacts during High Performance Liquid Chromatography peak fractionation processes

Therapeutic monoclonal antibodies (mAbs) are inherently heterogeneous and hence generally studied and controlled by an array of orthogonal separation methods. During drug candidate development, fractionation by HPLC is regularly employed to assist peak identification and product understanding. One overlooked challenge is the protein oxidation introduced by the fractionation process. In this study, we report the extent of fractionation-induced protein oxidation, which tends to complicate data interpretation and peak assignments. Higher-energy detectors such as fluorescence detectors and lower fraction concentration were found to exacerbate the oxidation artifacts. Other contributing factors than the detector-induced photostress were also found to contribute significantly to protein oxidation. Furthermore, our study showed that collecting fractions into a solution with oxidation scavengers, such as histidine and methionine, was effective in eliminating the oxidation artifacts introduced by detector exposure and fraction processing steps. Through an example, we demonstrate that the modified fractionation workflow improves the accuracy of peak assignments.

Luteinizing hormone (LH) formed a complex with an immunoglobulin G caused abnormally high levels of LH: A case report

We encountered a 30-year-old woman with remarkably elevated luteinizing hormone (LH) levels, as measured by electrochemiluminescent immunoassay (ECLIA), and no specific symptoms. We performed the following investigations: dilution linearity test, polyethylene glycol (PEG) precipitation test, immunoprecipitation test, protein G addition test, and high-performance liquid chromatography (HPLC) analysis. The linearity of patient’s serum was similar to that of a standard LH preparation, and non-specific reactions were not observed. The recovery rate of LH shown by the PEG precipitation test, immunoprecipitation test, and protein G addition test was low. Moreover, an abnormal peak in HPLC was located at a slightly larger molecular weight position than that of IgG. These results showed the presence of macro-LH, LH, and anti-LH-IgG autoantibody complex and suggested that the clearance of LH from the blood was delayed due to IgG binding, and therefore, the LH value was falsely high. We should keep the possibility of macro-LH in mind in cases of unexpectedly high LH values.

A Single Nucleotide Polymorphism in the SLCO1B3 Gene is Associated with Dose-Normalized Mycophenolic Acid AUC12

<h3>Purpose</h3> Lung transplant recipients are commonly maintained on mycophenolic acid (MPA). However, research has shown that transplant recipients experience a wide variability in MPA pharmacokinetics. We hypothesize that some of this variability is due to genetic factors. To this end we analyzed the relationship between MPA concentration and a hypofunctional polymorphism in the SLCO1B3 gene, rs7311358, which we have previously reported is associated with MPA-related clinical outcomes. <h3>Methods</h3> We performed full pharmacokinetic testing in 30 adult bilateral lung transplant recipients who were maintained on MPA and who had previously enrolled in our lung transplant genetic database. MPA levels were assessed using a high-performance liquid chromatography peak area quantitation. We calculated the 12-hour area under the curve (AUC₁₂) using the linear trapezoidal method. We used linear regression to evaluate the relationship between the SLCO1B3 polymorphism and the AUC₁₂. We also performed multivariate linear regression analysis to evaluate potential confounders. <h3>Results</h3> The SLCO1B3 rs7311358 polymorphism was present in 8 of 30 patients. These patients had a significantly lower dose normalized MPA AUC₁₂ than patients without the polymorphism (rs7311358 GG/GA 40.11 mg*h/L vs. rs7311358 AA 68.84 mg*h/L, p=0.041, Figure 1). Additionally, in multivariate testing the presence of the polymorphism remained significantly associated with MPA AUC₁₂. <h3>Conclusion</h3> We provide the first evidence that SLCO1B3 polymorphism rs7311358 is significantly associated with MPA dose normalized AUC₁₂ in lung recipients. Integration of this pharmacogenetic data with traditional pharmacokinetic testing could lead to more effective MPA management strategies.

Screening of Dry Blood Spots from Newborns by Two High Performance Liquid Chromatography (HPLC) Systems: A Comparison of Their Ability to Diagnose Both Sickle and Non-sickle Hemoglobinopathies.

Screening of newborns for the presence of sickle hemoglobin (HbS) is aimed at reducing the morbidity and mortality associated with sickle cell disease in early childhood. The high cost and limited availability of dedicated high performance liquid chromatography (HPLC) systems specially designed for screening of dry blood spots (DBS), however, restrict a wider application of this preventive approach. Therefore, we examined the ability of a commonly used HPLC system for detection of hemoglobinopathies in DBS samples in order to find an alternative for the dedicated newborn screening (NBS) HPLC system. DBS samples from 7522 newborns were first examined by Variant NBS HPLC system (Bio Rad, USA) for the presence of hemoglobinopathies. Positive samples were then analysed by Variant II system (Bio Rad, USA), another platform commonly used for hemoglobinopathy screening of anticoagulated blood samples. Eighty six newborns (1.1%) showed the presence of hemoglobinopathies (HbS 28, HbE 21, HbD 27, HbQ India 9 and Hb Barts 1) by Variant NBS system-all in heterozygous state. There was 100% correlation between the two sets of results obtained by the two HPLC systems. Newborns with HbQ India showed an additional Hb peak in HPLC resulting from combination of the abnormal alpha globin chain of HbQ India with the normal gamma chain of HbF-'HbF Q India'. Variant II HPLC system, used for routine hemoglobinopathy screening in anticoagulated blood, can also be used for screening DBS samples. This obviates the need for a dedicated NBS system for hemoglobinopathy screening in newborns. We also demonstrated that both the systems are equally competent in detecting non-sickle Hb variants in DBS samples.

Combining Field-Enabled Capillary Vibrating Sharp-Edge Spray Ionization with Microflow Liquid Chromatography and Mass Spectrometry to Enhance 'Omics Analyses.

Field-enabled capillary vibrating sharp-edge spray ionization (cVSSI) has been combined with high-flow liquid chromatography (LC) and mass spectrometry (MS) to establish current ionization capabilities for metabolomics and proteomics investigations. Comparisons are made between experiments employing cVSSI and a heated electrospray ionization probe representing the state-of-the-art in microflow LC-MS methods for 'omics studies. For metabolomics standards, cVSSI is shown to provide an ionization enhancement by factors of 4 ± 2 for both negative and positive ion mode analyses. For chymotryptic peptides, cVSSI is shown to provide an ionization enhancement by factors of 5 ± 2 and 2 ± 1 for negative and positive ion mode analyses, respectively. Slightly broader high-performance liquid chromatography peaks are observed in the cVSSI datasets, and several studies suggest that this results from a slightly decreased post-split flow rate. This may result from partial obstruction of the pulled-tip emitter over time. Such a challenge can be remedied with the use of LC pumps that operate in the 10 to 100 μL·min-1 flow regime. At this early stage, the proof-of-principle studies already show ion signal advantages over state-of-the-art electrospray ionization (ESI) for a wide variety of analytes in both positive and negative ion mode. Overall, this represents a ∼20-50-fold improvement over the first demonstration of LC-MS analyses by voltage-free cVSSI. Separate comparisons of the ion abundances of compounds eluting under identical solvent conditions reveal ionization efficiency differences between cVSSI and ESI and may suggest varied contributions to ionization from different physicochemical properties of the compounds. Future investigations of parameters that could further increase ionization gains in negative and positive ion mode analyses with the use of cVSSI are briefly presented.

Quality control method of sterols in fermented Cordyceps sinensis based on combined fingerprint and quantitative analysis of multicomponents by single marker.

In this study, we established a new pattern for differentiating and comprehensively evaluating the quality of fermented Cordyceps sinensis based on high-performance liquid chromatography (HPLC) fingerprint analysis combined with similar analysis (SA), principal component analysis (PCA), hierarchical cluster analysis (HCA), and the quantitative analysis of multicomponents by single marker (QAMS). These methods indicated that fermented Cordyceps sinensis samples could be categorized into one class by PCA and HCA. The fingerprints of fermented Cordyceps sinensis were established, and four HPLC peaks were identified as ergosterol, daucosterol, stigmasterol, and β-sitosterol in Jinshuibao capsules and tablets (two products of fermented Cordyceps sinensis). Ergosterol was chosen as the internal reference substance, and the relative correction factors (RCFs) between ergosterol and the other three sterols were calculated using the QAMS method. Moreover, the accuracy of the QAMS method was confirmed by comparing the relative error between the results of the method used with those of an external standard method (ESM). No significant difference between the two methods was observed. The total sterols content in Jinshuibao products were calculated by the QAMS method, and the total sterols content of the two products were similar. This study showed that the method established herein was efficient and successful in the identification fermented Cordyceps sinensis and may further act to facilitate systematic quality control of fermented Cordyceps sinensis products.

Identification of tuliposide G, a novel glucoside ester-type tuliposide, and its distribution in tulip.

Tuliposides (Pos) are major defensive secondary metabolites in tulip (genus Tulipa), having 4-hydroxy-2-methylenebutanoyl and/or (3S)-3,4-dihydroxy-2-methylenebutanoyl groups at the C-1 and/or C-6 positions of d-glucose. The acyl group at the C-6 position is converted to antimicrobial lactones, tulipalins, by tuliposide-converting enzymes (TCEs). In the course of a survey of tulip tissue extracts to identify novel Pos, we found a minute high-performance liquid chromatography peak that disappeared following the action of a TCE, and whose retention time differed from those of known Pos. Spectroscopic analyses of the purified compound, as well as its enzymatic degradation products, revealed its structure as 5″-O-(6-O-(4'-hydroxy-2'-methylenebutanoyl))-β-d-glucopyranosyl-(2″R)-2″-hydroxymethyl-4″-butyrolactone, which is a novel glucoside ester-type Pos. We gave this compound the trivial name 'tuliposide G' (PosG). PosG accumulated in bulbs, at markedly lower levels than 6-PosA (the major Pos in bulbs), but was not found in any other tissues. Quantification of PosG in bulbs of 52 types of tulip, including 30 cultivars (Tulipa gesneriana) and 22 wild Tulipa spp., resulted in the detection of PosG in 28 cultivars, while PosG was present only in three wild species belonging to the subgenus Tulipa, the same subgenus to which tulip cultivars belong, suggesting the potential usefulness of PosG as a chemotaxonomic marker in tulip.

Aligning retention time shifts in HPLC three-dimensional spectra by icoshift approach combined with data arrangement methods and the release of a graphical user interface.

High-performance liquid chromatography coupled with photodiode array detection has been extensively applied in many fields and the peaks among the analyzed samples can be shifted due to the variations of instrumental and experimental conditions. In multivariate analysis, retention time alignment is an important pretreatment step. Hence, the shifted peaks in high-performance liquid chromatography coupled with photodiode array detection three-dimensional spectra should be aligned for further analysis. Being motivated by this purpose, the interval correlated shifting method combined with the proposed data arrangement methods are recommended and employed on high-performance liquid chromatography coupled with photodiode array detection data as a demonstration. We validate the alignment performance of the proposed method through comparison the consistency of the retention time before and after alignment. The obtained results demonstrated that the proposed method is capable of successful aligning the employed data. Additionally, the interval correlated shifting method combined with the data arrangement modes is implemented in an easy-to-use graphical user interface environment and so can be operated easily by users not familiar with programming languages.

Isolation and identification of tuliposides D and F from tulip cultivars.

6-Tuliposides A (6-PosA) and B (6-PosB) are major defensive secondary metabolites in tulip cultivars (Tulipa gesneriana), having an acyl group at the C-6 position of d-glucose. Although some wild tulip species produce 1,6-diacyl-glucose type of Pos (PosD and PosF), as well as 6-PosA/B, they have not yet been isolated from tulip cultivars. Here, aiming at verifying the presence of PosD and PosF in tulip cultivars, tissue extracts of 25 cultivars were analyzed by high-performance liquid chromatography (HPLC). Although no HPLC peaks for PosD nor PosF were detected in most cultivars, we found two cultivars giving a minute HPLC peak for PosD and the other two cultivars giving that for PosF. PosD and PosF were then purified from petals of cultivar 'Orca' and from pistils of cultivar 'Murasakizuisho', respectively, and their identities were verified by spectroscopic analyses. This is the first report that substantiates the presence of 1,6-diacyl-glucose type of Pos in tulip cultivars.

Quadrupole-Time-of-Flight Mass Spectrometric Identification of Hemoglobin Subunits α, β, γ and δ in Unknown Peaks of High Performance Liquid Chromatography of Hemoglobin in β-Thalassemias

This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, α, β, δ and γ peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with β-thalassemia (β-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The α-, β-, δ- and γ-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with β-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1 min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the protein-aggregation disorders will require review of the role of free globin in the pathology of the disease.

Anti-oxidative and anti-proliferative activities of extracted phytochemical compound thymoquinone

To evaluate the anti-oxidant and anti-proliferative potential of Thymoquinone extracted from the essential oil of indigenous herbs of Nigella sativa and Thymus vulgaris. Extraction and quantification of Thymoquinone was carried out in July, 2017 in Department of Environmental Science, Lahore College for Women University (LCWU), Lahore. Thymoquinone was extracted from seeds of Nigella Sativa and aerial parts of Thymus vulgaris by employing soxhlet extraction with 1:4 ratios of nhexane and methanol. High Performance Liquid Chromatography was used to quantify Thymoquinone from the methanolic extracted oil of sample by applying calibration curve method. Extracted Thymoquinone was identified by sample peaks obtained at retention time were compared with peak of standard Thymoquinone at respective time. The Thymoquinone obtained from both samples was then subjected to Fourier-transform infrared spectroscopy for confirmation by identifying its functional groups. Anti-oxidant activities of both samples were measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) assay in Department of Environmental Science, LCWU. In-vitro anti-proliferative activities of extracted Thymoquinone were evaluated in HeLa cell cancer lines by cell proliferations Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay in Department of Microbiology, University of Veterinary and Animal Sciences (UVAS), Lahore. SPSS 18 and Graph pad prism 18 was used for data analysis. Soxhlet extraction with solvents ratios yielded 48.92% oil from Nigella sativa and 23.2 % from Thymus vulgaris. High Performance Liquid Chromatography peak of standard Thymoquinone was measured at retention time of 5.5 min which was then compared with the peak obtained from both samples at the similar retention time. The extracted Thymoquinone from both samples were quantified by calibration curve method showing 614.25 mg/L from Nigella sativa and 548.86 mg/L from Thymus vulgaris. The two anti-oxidant assays of both samples compared with standard Thymoquinone showed significant scavenging activities in dose amount manner. Cell proliferation of HeLa cancer significantly decreased with dose response manner (p<0.01), showing highest cell death in high concentration of Thymoquinone. Inhibitory concentration 50 (IC50) of cancer cell line treated with Nigella sativa oil was 0.5 μM and Thymus vulgaris was 18 μM compared to standard Thymoquinone, showing Inhibitory concentration50 (IC50) of 6 μM using Graph pad prism v.8.0. Both Nigella sativa and Thymus vulgaris were found to be the best source of Thymoquinone as chemotherapeutic drug expressed potent anti-oxidant and anti-proliferative activities.

Observation of the thermal influenced quantum behaviour of water near a solid interface

Water is essential for life. However, the structure and properties of water are still not well understood. It has been introduced that anomalies are in vicinal water near solid interfaces. We performed capillary flow experiments on water with a silica colloid sample using a high-performance liquid chromatography (HPLC) system by accurately varying the temperature and analysed the peak shape rigorously. We obtained a novel anomalous temperature spectrum from the peak-shape analysis. Here we report the observed distinct specific anomalous temperature (SAT) behaviour in vicinal water at silica interface. The anomaly appeared in the viscous force that was derived from a relationship between the shape of the HPLC peak and the velocity profile for the capillary flow. The observations were highly reproducible, and we conclude that the SAT is related to the quantum mechanical behaviour of water, in agreement of the characteristic acceptance of thermal displacement according to the Franck-Condon principle. We performed the same experiments using heavy water and water mixed with a small amount of methanol, and the results support the quantum phenomenological origin.