Coulometric Electrochemical Detection Research Articles

Vitamin C Urinary Loss and Deficiency in Human Immunodeficiency Virus (HIV): Cross-sectional Study of Vitamin C Renal Leak in Women With HIV.

Reduced plasma vitamin C (vitC) concentrations in human immunodeficiency virus (HIV) may result from abnormal urinary excretion: a renal leak. VitC renal leak indicates underlying nutritional dysregulation independent of diet. We hypothesized that increased renal leak prevalence in HIV would be associated with deficient vitC concentrations. We conducted an outpatient cross-sectional study of 96 women (40 HIV [PWH] and 56 without HIV [PWOH]) at the National Institutes of Health and Georgetown University. Renal leak was defined as abnormal urinary vitC excretion at fasting plasma concentrations <43.2µM, 2 SDs below vitC renal threshold in healthy women. To determine the primary outcome of renal leak prevalence, matched urine and plasma samples were collected the morning after overnight fast. Secondary outcomes assessed group differences in mean plasma vitC concentrations and prevalence of vitC deficiency. Exploratory outcomes assessed clinical parameters associated with renal leak. VitC was measured by high-performance liquid chromatography with coulometric electrochemical detection. PWH had significantly higher renal leak prevalence (73%vs14%; OR (odds ratio):16; P<.001), lower mean plasma vitC concentrations (14µMvs50µM; P<.001), and higher prevalence of vitC deficiency (43%vs7%; OR:10; P<.001) compared with PWOH, unchanged by adjustments for confounding factors. Significant predictors of renal leak included antiretroviral therapy (ART), Black race, older age, and metabolic comorbidities but not viral load or CD4 count. When compared with other chronic disease cohorts, PWH had the highest prevalence of renal leak and vitC deficiency (P<.001). High prevalence of vitC renal leak in HIV was associated with vitC deficiency, ART use, and race/ethnicity differences.

PSUN168 The relationship between vitamin C concentrations and reduced red blood cell deformability in diabetes

Abstract Background Microvascular angiopathy is a common complication for patients with diabetes. Reduced red blood cell (RBC) deformability in diabetes has been associated with micro- and macrovascular complications such as diabetic nephropathy and coronary artery disease. Preliminary studies in mice have shown an inverse relationship between RBC vitamin C concentrations and RBC deformability measures. This study investigated the relationship between RBC vitamin C concentration and cell deformability measures. Methods We analyzed RBC deformability and vitamin C level in a cross-sectional cohort study of individuals with diabetes (n=82) and non-diabetic controls (n=80). Subjects were studied outpatient, with samples obtained following an overnight fast. RBC rigidity parameters defined as SS1/2 (shear stress at which half of the maximum deformability occurs) was assessed using the laser diffraction viscometry ektacytometer (LORRCA, Mechatronics, Netherland). We also quantified osmotic sensitivity by measuring deformability under an osmotic gradient (osmoScan, o-hyper; osmolality corresponding to 50% of the Elmax). Vitamin C in plasma was measured using coulometric electrochemical detection with high performance liquid chromatography. We evaluated the association between these deformability parameters, vitamin C concentration. Results Compared to the non-diabetic cohort, the diabetes cohort had significantly lower RBC vitamin C concentration, respectively 28.1µM and 42.9µM (p=0.0013). Low RBC vitamin C concentration was significantly associated with reduce RBC deformability measured by increased SS1/2 (p=0.009). Additionally, low RBC vitamin C was associated with decreased o-hyper, suggesting a decrease dehydration of RBC (p=0.0250). Conclusion Our data demonstrate reduced vitamin c levels in the diabetic cohort. Reduced RBC deformability is associated with reduced RBC vitamin C concentration. Thus, reduced vitamin C levels in diabetic patients might increase red blood cell rigidity and reduce deformability. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

PSUN169 Vitamin C Renal Leak in Diabetes

Abstract Background People with diabetes have lower plasma vitamin C concentrations compared to nondiabetic individuals. The prevalence of urinary vitamin C loss, and its relationship to plasma vitamin C concentrations are unknown. Methods In this cross-sectional cohort study of 162 individuals; 82 with diabetes and 80 nondiabetic controls, we investigated the prevalence and clinical characteristics of vitamin C renal leak, using fasting plasma and matched 1h urine samples. Based on vitamin C renal threshold, vitamin C renal leak was defined as presence of vitamin C in urine with plasma concentrations less than the minimal elimination threshold (MET) of 43.2µM in women or 38.1µM in men. We also assessed group differences in mean plasma vitamin C concentrations and investigated the association between diabetes-related clinical variables and renal leak. Vitamin C in plasma and urine was measured using coulometric electrochemical detection with high performance liquid chromatography. Results When compared with nondiabetic controls, subjects with diabetes had significantly increased prevalence of vitamin C renal leak (8.8% vs 32.9%: OR5, p&amp;lt;0.001) and decreased plasma vitamin C concentrations (53.1µM vs 40.9µM, p&amp;lt;0.001). Higher fasting plasma glucose, glycosylated hemoglobin A1c, body mass index, micro/macro vascular complications and protein creatinine ratio were predictive of vitamin C renal leak. Conclusion Increased prevalence of vitamin C renal leak in diabetes is associated with reduced plasma vitamin C concentrations. Glycemic control, microvascular complications, obesity, and proteinuria were associated with renal leak. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

The new rapid and accurate analytical HPLC‐ECD method for the determination of rosmarinic acid in meat products

A new rapid method has been developed for the determination of low levels of rosmarinic acid extracted from rosemary (Rosmarinus officinalis L.) and has been used as an antioxidant in meat and meat products after cold storage at 4°C. The method is a high performance liquid chromatography using a coulometric electrochemical detector. It provides a significant improvement on the limit of detection, which was 0.33 ppb, while the limit of quantification was 1 ppb of rosmarinic acid. The advantage of the method also lies in the simpler and faster sample preparation, which can quantify a very low concentration of rosmarinic acid (60 ppb), and is more than 40 ppb below the limits of previously existing methods. A coulometric method is well suited for determining low analyte concentrations and is one of the most sensitive analytical approaches available today, in addition to being time efficient and cost effective. PRACTICAL APPLICATION: A new method for determining low concentrations (60 ppb) of rosmarinic acid in meat and meat products is presented. The method is user-friendly, as it does not require complex sample preparation. It is a selective, precise, and accurate method that makes it useful for routine applications in the meat and other food industries.

HPLC electrochemical detection and quantification of monoamines and their metabolites in rat brain tissue samples.

The monitoring of monoamines and their metabolites in CNS samples can be very valuable in pharmaceutical and biomedical research. A specific high performance liquid chromatography, coupled to a coulometric electrochemical detection method, for the assay of monoamines (dopamine, norepinephrine, epinephrine and serotonin) and their metabolites in rat brain tissue samples was developed. The chromatographic separation was achieved on a C8 reversed phase column with a mobile phase consisting of 0.1 M sodium formate buffer, 5 mM sodium 1-heptanesulfonate, 0.17 mM ethylenediaminetetraacetic acid disodium salt and 5% v/v acetonitrile (pH ±4.0). The detection was achieved through electrochemical detection, with a coulometric cell potential setting of +650 mV. The flow-rate was at 1 ml/min and the total run time was 50 min. The method was validated according to validation guidelines. The method was found to be linear (R2 > 0.99) over the analytical range (5 to 200 ng/ml) for all monoamines and their metabolites. All the other validation parameters were acceptable and within range. The method was applied to three rat brain areas (pre-frontal cortex, hippocampus and striatum), where the monoamines (except for epinephrine) and their metabolites were easily detected.

Analysis of human cerebrospinal fluid monoamines and their cofactors by HPLC.

The presence of monoamines and their cofactors (the pterins and vitamin B6 (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors. This protocol describes step-by-step procedures for CSF sample preparation for the analysis of different molecules, HPLC calibration and analysis, and data quantification and interpretation. Unlike plasma/tissue/blood samples, CSF requires minimal sample preparation: in this protocol, only the analysis of PLP requires mixing with trichloroacetic acid to release the protein-bound vitamin, centrifugation, and mixing of the supernatant with phosphate buffer and sodium cyanide for derivatization in alkaline conditions. Monoamines are analyzed by HPLC with coulometric electrochemical detection (ED), pterins are analyzed by HPLC with coupled coulometric electrochemical and fluorescence detection, and PLP is analyzed by HPLC with fluorescence detection. The quantification of all compounds is achieved by external calibration procedures, and internal quality control and standards are analyzed in each run. We anticipate that investigation of dopamine and serotonin disturbances will be facilitated by measurements of these compounds in human CSF and other biological samples. The estimated time for the different procedures primarily depends on the electrochemical detector stabilization. Overnight stabilization of this detector is advised, and, after that step, preanalytical equilibration rarely exceeds 3 h.

Detection of B6 Vitamers in Grain Products: Experimental and Computational Studies

A reversed phase high-performance liquid chromatography method (HPLC) with coulometric electrochemical detection has been applied and validated for the simultaneous analysis of pyridoxamine (PM), pyridoxal (PL), and pyridoxine (PN) in cereal products. Isocratic separation was achieved using a C18 column with a mobile phase consisting of methanol-phosphate buffer (10:90) and 0.018 M trimethylamine adjusted to pH 3.55 with 85% orthophosphoric acid. The limits of detection for PM, PL, and PN were 0.28, 0.36, and 0.43 ng mL−1, respectively, with vitamins recoveries ranging from 90.4 to 98.1%. The applied method for the analysis of B6 vitamins naturally present in grain products, offers a simple and fast sample preparation without derivatization. To understand vitamer separation further, methods of computational chemistry were employed. Specifically, density functional theory (DFT) was used to determine electrostatic potentials, as well as the charges of each vitamer. A number of correlations were established between these properties and elution order.

Relevance of plasma levels of free homocysteine and methionine as risk predictors for ischemic stroke in the young

The debated vascular risk potential of total homocysteine (tHcy), due to failed clinical trials designed on B vitamin supplementation, raises many possible explanations like the higher risk potential of the deleterious, free form of homocysteine (fHcy) or, the unchecked confounding effects of B-vitamins in tHcy-based association studies. Additionally, the cardiovascular risk probability of altered status of the homocysteine precursor, methionine (tMet) could shed light on the causality of association between tHcy and cardiovascular diseases. Hence, we aimed to evaluate the risk associations of elevated plasma levels of tHcy, fHcy and low levels of tMet with premature, ischemic stroke. We recruited 171 young, ischemic stroke patients (aged ≤45 years) and 249 age- and gender-matched healthy controls. Plasma levels of fHcy, tHcy, tMet and vitamin B6 were estimated using HPLC coupled with coulometric electrochemical detection. Plasma levels of vitamin B12 and folate were estimated by radioimmunoassay. Elevated fHcy (>2.9μmol/L) was independently and strongly associated with the risk of premature, ischemic stroke (OR=9.62, 95% CI=3.51-26.40). On the contrary, association between premature ischemic stroke and elevated tHcy (>15.0μmol/L) was found to attenuate when adjusted for vitamin B6 values (OR=0.24, 95%, CI=0.03-1.69). Interestingly, compromised B6-status (<59.2nmol/l) was found to confer high risk of premature ischemic stroke (OR=170.80, 95% CI=58.22-501.06). We could not establish any significant correlation between fHcy and B-vitamin levels (P>0.05). Low tMet (<13.86μmol/L) was also not significantly associated with premature, ischemic stroke (OR=2.53, 95%CI=0.613-10.38). Our results indicate significant but not-correlated, independent associations of fHcy and vitamin B6 with risk of premature, ischemic stroke. However, the causality of these associations need prospective and large scale validations. Further, our findings highlight the crucial confounding effects of B-vitamins on risk association between tHcy and premature ischemic stroke.

Plasma S-adenosylhomocysteine: A potential risk marker for cerebral venous thrombosis

BackgroundDespite a plethora of studies suggesting that hyperhomocysteinemia is associated with an increased risk for arterial and venous thrombosis, there is paucity of data on the role of the S-adenosylhomocysteine (SAH), the metabolic precursor of homocysteine (Hcy) as a risk predictor for cerebral venous thrombosis (CVT). MethodWe estimated fasting plasma concentrations of total homocysteine (tHcy), SAH and S-adenosylmethionine (SAM), in 185 CVT patients and 248 healthy controls, by reverse-phase high performance liquid chromatography coupled with coulometric electrochemical detection. ResultsFasting tHcy, SAH and SAM were significantly higher in patients compared with controls. Increased tHcy and SAH concentrations were associated with 4.54-fold (95% CI, 2.74–7.53) and 35.77-fold (95% CI, 19.45–65.79) increase in risk for CVT, respectively. Receiver operating characteristic (ROC) curve analysis showed that the area under curve, sensitivity and specificity was higher for SAH compared to tHcy. Further, discriminant analysis to distinguish between tHcy and SAH showed that SAH had a significantly higher percentage classification, with lower Wilk's lambda and higher χ2, compared to tHcy. ConclusionIncreased plasma SAH may be a more sensitive risk marker for CVT than plasma tHcy.

Prevalence of β(S)-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil.

The aim of this study was to determine the frequency of beta S-globin gene (βS globin) haplotypes and alpha thalassemia with 3.7 kb deletion (−α3.7kb thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of βS globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The βSglobin haplotypes and −α3.7kb thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the −α3.7kb mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity.

Biomarker differences between cadaveric grafts used in human orthotopic liver transplantation as identified by coulometric electrochemical array detection (CEAD) metabolomics.

Metabolomics in systems biology research unravels intracellular metabolic changes by high throughput methods, but such studies focusing on liver transplantation (LT) are limited. Microdialysate samples of liver grafts from donors after circulatory death (DCD; n=13) and brain death (DBD; n=27) during cold storage and post-reperfusion phase were analyzed through coulometric electrochemical array detection (CEAD) for identification of key metabolomics changes. Metabolite peak differences between the graft types at cold phase, post-reperfusion trends, and in failed allografts, were identified against reference chromatograms. In the cold phase, xanthine, uric acid, and kynurenine were overexpressed in DCD by 3-fold, and 3-nitrotyrosine (3-NT) and 4-hydroxy-3-methoxymandelic acid (HMMA) in DBD by 2-fold (p<0.05). In both grafts, homovanillic acid and methionine increased by 20%-30% with each 100 min increase in cold ischemia time (p<0.05). Uric acid expression was significantly different in DCD post-reperfusion. Failed allografts had overexpression of reduced glutathione and kynurenine (cold phase) and xanthine (post-reperfusion) (p<0.05). This differential expression of metabolites between graft types is a novel finding, meanwhile identification of overexpression of kynurenine in DCD grafts and in failed allografts is unique. Further studies should examine kynurenine as a potential biomarker predicting graft function, its causation, and actions on subsequent clinical outcomes.

Determination of A-type and B-type procyanidins in apple, cocoa and cinnamon extracts

Flavan-3-ols, known also as proanthocyanidins or condensed tannins, after lignans are the second most common class of natural phenolic substances found in nature. Single linked, B-type procyanidins occur in many foods, with those from apples and cocoa being the most prominent in western diet. A possible connection between procyanidins and cardiovascular disease is particularly interesting. The double linked, A-type procyanidins occur in cinnamon and cranberries and are believed to contribute to anti-diabetic and anti-UTI properties of these foods. Procyanidins form chains containing polymerized subunits of epicatechin and catechin, with the number of subunits being between 2 and 30. With the ever increasing interest in determining their significance as dietary antioxidants and pharmacologically active components, better analytical techniques allowing their standardization in dietary supplements are required. We developed a sensitive and selective gradient HPLC method combining photodiode array and coulometric electrochemical array detection to resolve and quantify numerous procyanidins in different foods/supplements. This approach also enabled the estimation of standard purity and abundance of contaminants. A second method, using charged aerosol detection, was used to determine the presence of impurities that either lack a chromophore or are not electrochemically active. A third method was developed using gradient UHPLC with UV detection. This approach significantly shortened the analysis time from 30 to < 5 minutes, while maintaining analyte resolution.

The Use of Gradient HPLC with Coulometric Electrochemical Array Detection and Pattern Recognition to Study Botanical Authenticity

Although the authenticity and adulteration of botanicals continue to be major issues, there are relatively few simple methods available for the screening of large numbers of commercial batches of product. The challenge arises from the complexity and variability of genuine material combined with the unrelenting conduct of adulteration. Variety, growing region, season, ripeness, and processing methods all contribute to the variability of the authentic product, making unambiguous characterization difficult.

Synthesis, HPLC Measurement and Bioavailability of the Phenolic Amide Amkamide

Amkamide is a phenolic amide whose analogues were recently reported to have potent mitochondria protective activity, although their bioavailability is still unknown. Therefore, in this study, amkamide was synthesized and confirmed by nuclear magnetic resonance, and a high-performance liquid chromatography (HPLC) method was developed for analyzing the amides in biological samples. HPLC separation was performed on a Nova-Pak C18 column using a gradient condition and a coulometric electrochemical detector. The HPLC method was able to produce excellent and reproducible separations of amkamide from other amides (oretamide, becatamide, enferamide and veskamide). All five amides yielded outstanding peak resolutions with detection limits as low as 100 fmol. Therefore, the bioavailability of amkamide together with becatamide was determined in a mouse model. Their plasma concentrations were measured following two oral administrations (2 and 4 mg per 30 g body weight) using the HPLC method. As expected, relatively high amounts of amkamide and becatamide (51-58 µM) were detected at the time after administration when the maximum concentration was reached (approximately 20 and 25 min) at the high dose. The data indicate that HPLC methods with amide standards may be utilized to measure amkamide and its analogues in biological samples with excellent resolution and detection limits.

A validated method for the determination of selected phenolics in olive oil using high-performance liquid chromatography with coulometric electrochemical detection and a fused-core column

A liquid chromatographic method with a coulometric electrochemical detector (ECD) and a fused-core column was developed for the quantification of the olive oil phenolics tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acid. The method was validated according to guidelines of the U.S. Food and Drug Administration. The selectivity, linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, as well as the stabilities of the phenolic standards and quality control samples were determined. The separation of the eight phenolic compounds was achieved within 16min and the total analysis time (35min) was ca. 3-fold shorter than that of conventional HPLC methods. The LOQ range was 0.3–15.3ng/mL, which is at least 5-fold lower than those of other methods. Recovery was between 75% and 101%. Overall the method has the advantages of being sensitive, selective, fast and provides simultaneous qualitative and quantitative analysis of phenolics.

Comprehensive Analysis of Polyphenols in 55 Extra Virgin Olive Oils by HPLC-ECD and Their Correlation with Antioxidant Activities

In this study, we analyzed eight phenolic compounds (tyrosol, hydroxytyrosol, oleuropein, pinoresinol, and caffeic, ferulic, vanillic, and p-coumaric acid) in 55 mono- and multivarietal extra virgin olive oil samples by high performance liquid chromatography (HPLC) coupled to a coulometric electrochemical array detector (ECD). The phenolic profile of olive oil samples differed depending on the geographical origin and olive variety. The total reducing capacity (total phenolics) of olive oils ranged from about 40 to 530 mg gallic acid equivalents/kg oil. Tyrosol, hydroxytyrosol and pinoresinol were the most abundant phenolic compounds in olive oils. The antioxidant capacity of the olive oil extracts was determined by ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays. Total reducing capacity was significatly correlated with FRAP (R² = 0.91, p < 0.001) and TEAC (R² = 0.92, p < 0.001) values. Total reducing capacity, TEAC and FRAP values were significantly correlated with tyrosol, hydroxytyrosol as well as oleuropein concentrations. Hydroxytyrosol, comprising over 40 % of total olive oil phenolics, mainly contributed to the antioxidant activity of olive oils. The present study provides a comprehensive database of polyphenols in olive oils from 9 different countries and four continents.

An in vitro study on the pharmacological ascorbate treatment of influenza virus

To investigate the effects and mechanism of pharmacological ascorbate against Influenza A/CA/7/09 (H1N12009). NHBE cells (≈ 95% confluent monolayer) in 12-well plates (Corning) were kept at 37°C at all times. NHBE cells were exposed to A/CA/7/09 (H1N12009) influenza virus at MOI of 0.01 for 1 h, rinsed with NHBE medium, and incubated with NHBE medium containing 20 mmol/L ascorbate or 20 mmol/L ascorbate +600 IU/ml Catalase. The cells were then incubated for an additional 4 - 12 h and the culture medium was harvested for titration. Viral titers were determined as log(10) 50% tissue culture infective doses (TCID₅₀) assay in MDCK cells. Ascorbate in NHBE medium was determined using HPLC separation coupled with coulometric electrochemical detection. Hydrogen peroxide was detected indirectly by Clark-type oxygen electrode. In vitro experiments showed that pharmacological ascorbate killed not only isolated viruses, but also viruses from normal human bronchial epithelial cells. The antiviral effect of ascorbic acid appeared to be dose-dependent. 2.5 mmol/L ascorbic acid was able to eliminate 90% of the viruses and 20 mmol/L ascorbic acid totally blocked viral replication in vitro. The antiviral effect of pharmacological ascorbate varied at different phases of infection. Pharmacological ascorbate eliminated viral infectivity with treatment times as short as 4 hours at early stage of infection. But the effect was reversed by catalase. Pharmacological ascorbate (vitamin C) as a pro-drug eliminates or kills influenza virus, probable by producing steady-state concentrations of hydrogen peroxide (H₂O₂) in extracellular fluid.

Vitamin C in mouse and human red blood cells: An HPLC assay

Although vitamin C (ascorbate) is present in whole blood, measurements in red blood cells (RBCs) are problematic because of interference, instability, limited sensitivity, and sample volume requirements. We describe a new technique using HPLC with coulometric electrochemical detection for ascorbate measurement in RBCs of humans, wild-type mice, and mice unable to synthesize ascorbate. Exogenously added ascorbate was fully recovered even when endogenous RBC ascorbate was below the detection threshold (25nM). Twenty microliters of whole blood or 10μl of packed RBCs was sufficient for assay. RBC ascorbate was stable for 24h from whole-blood samples at 4°C. Processed, stored samples were stable for >1month at −80°C. Unlike other tissues, ascorbate concentrations in human and mouse RBCs were linear in relation to plasma concentrations (R=0.8 and 0.9, respectively). In healthy humans, RBC ascorbate concentrations were 9–57μM, corresponding to ascorbate plasma concentrations of 15–90μM. Mouse data were similar. In human blood stored as if for transfusion, initial RBC ascorbate concentrations varied approximately sevenfold and decreased 50% after 6weeks of storage under clinical conditions. With this assay, it becomes possible for the first time to characterize ascorbate function in relation to endogenous concentrations in RBCs.

The Spectro-Electro Array: A Novel Platform for the Measurement of Secondary Metabolites in Botanicals, Supplements, Foods and Beverages.

Plants contain an amazingly diverse range of secondary metabolites, many of which are purported to offer health benefits. The challenge for the analytical chemist for the measurement of these compounds is two-fold: first, to develop assays that can accurately discriminate between compounds that often have similar physicochemical characteristics; second, to analyze such compounds in complicated matrices including botanicals, supplements, foods and beverages. Gradient HPLC with diode array detection (DAD) is often used for the determination of natural products. However, this approach often suffers from a lack of specificity as compounds with similar structures may co-elute chromatographically and cannot be deconvoluted spectrally. Furthermore, this technique lacks sensitivity limiting its use for the study of natural product metabolism in animals and humans. Coulometric electrochemical array detection, on the other hand, is selective, being able to distinguish between subtle changes in chemical structure and, with sub picogram limits of detection, can be used to examine natural product absorption and metabolism. The combination of DAD and coulometric electrochemical array detection extends the range of compounds measured by either technology alone. The technique has been used to resolve and quantify specific phytochemicals in crude extracts of a variety of natural products supplements (e.g., ginseng, black cohosh and ginko), beverages (black and green tea), culinary herbs (oregano, rosemary, sage and thyme) and spices (clove and nutmeg). In addition to quantitative analyses, changes in the pattern of metabolites, when evaluated using chemometric modeling software, can be used to study: product adulteration, contamination, composition and stability and in the case of wine and fruit juice, the effect of growing region and differences between varietals.

The Application of HPLC with Coulometric Electrochemical Array Detection to the Study of Natural Products and Botanicals: From Targeted Analyses to Metabolomics

Many potentially important bioactive phytochemicals are electrochemically active and can be measured using HPLC with electrochemical detection (HPLC-ECD). Such compounds, come from diverse chemical classes and include phenols, polyphenols, aromatic amines, quinones, isoquinolines, indoles, thiols and conjugated polyenes. Although HPLC-ECD with a single amperometric working electrode offers the analyst excellent sensitivity and some degree of selectivity, this approach is limited as it cannot be used routinely with gradient chromatography, relatively few analytes are measured concurrently, and qualitative information is lacking. The coulometric flow-through graphite working electrode is both sensitive (˜100% of an analyte will react) and maintenance free. When used in series, chromatographically co-eluting analytes can be resolved voltammetrically. Such behavior can be used to identify and characterize an analyte in an analogous fashion to the use of spectral data from a diode array detector. Furthermore, the CoulArray® detector, with its array of up to sixteen serially placed coulometric sensors, is fully gradient compatible and can be used for either targeted or metabolomics studies. During this presentation I will illustrate the capability of coulometric array detection for the measurement of specific analytes in plant, animals and human tissues. I will also discuss the use of metabolite profiles/metabolomics with pattern recognition and how this can be used to study product and botanical adulteration, contamination and composition.