Quantitative determination of potential urine biomarkers of respiratory illnesses using new targeted metabolomic approach

Western Economic Diversification Canada, AllerGen NCE, Saskatchewan Health Research FoundationPeer ReviewedThe diagnosis of asthma and chronic obstructive pulmonary disease (COPD) can be challenging due to the overlap in their clinical presentations in some patients. There is a need for a more objective clinical test that can be routinely used in primary care settings. Through an untargeted 1H NMR urine metabolomic approach, we identified a set of endogenous metabolites as potential biomarkers for the differentiation of asthma and COPD. A subset of these potential biomarkers contains 7 highly polar metabolites of diverse physicochemical properties. To the best of our knowledge, there is no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that evaluated more than two of the target metabolites in a single analytical run. The target metabolites belong to the families of monosaccharides, organic acids, amino acids, quaternary ammonium compounds and nucleic acids, rendering hydrophilic interaction liquid chromatography (HILIC) an ideal technology for their quantification. Since a clinical decision is to be made from patients data, a fully validated analytical method is required for biomarker validation. Method validation for endogenous metabolites is a daunting task since current guidelines were designed for exogenous compounds. As such, innovative approaches were adopted to meet the validation requirements. Herein, we describe a sensitive HILIC-MS/MS method for the quantification of the 7 endogenous urinary metabolites. Detection was achieved in the multiple reaction monitoring (MRM) mode with polarity switching, using quadrupole-linear ion trap instrument (QTRAP 6500) as well as single ion monitoring in the negative-ion mode. The method was fully validated according to the regulatory guidelines. Linearity was established between 6 and 21000 ng/mL and quality control samples demonstrated acceptable intra- and inter-day accuracy (85.7%-112%), intra- and inter-day precision (CV% <11.5%) as well as stability under various storage and sample processing conditions. To illustrate the method's applicability, the validated method was applied to the analysis of a small set of urine samples collected from asthma and COPD patients. Preliminary modelling of separation was generated using partial least square discriminant analysis (R2 0.752 and Q2 0.57). The adequate separation between patient samples confirms the diagnostic potential of these target metabolites as a proof-of-concept for the differentiation between asthma and COPD. However, more patient urine samples are needed in order to increase the statistical power of the analytical model

Comparison of accuracy and precision between multipoint calibration, single point calibration and relative quantification for targeted metabolomic analysis

Natural Sciences and Engineering Research Council of Canada (NSERC); Western Economic Diversification CanadaPeer ReviewedTargeted metabolomics requires accurate and precise quantification of candidate biomarkers, often through tandem mass spectrometric (MS/MS) analysis. Differential isotope labeling (DIL) improves mass spectrometric (MS) analysis in metabolomics by derivatizing metabolites with two isotopic forms of the same reagent. Despite its advantages, DIL-liquid chromatographic (LC)-MS/MS can result in substantial increase in workload when fully validated quantitative methods are required. To decrease the workload, we hypothesized that single point calibration or relative quantification could be used as alternative methods. Either approach will result in significant saving in resources and time. To test our hypothesis, six urinary metabolites were selected as model compounds. Urine samples were analyzed using a fully-validated multipoint dansyl chloride-DIL-LC-MS/MS method. Samples were reprocessed using single point calibration and relative quantification modes. Our results demonstrated that the performance of single point calibration or relative quantification was inferior, for some metabolites, to multipoint calibration. The lower limit of quantification failed in the quantification of ethanolamine in most of participant samples using single point calibration. In addition, its precision was not acceptable in one participant during serine and ethanolamine quantification. On the other hand, relative quantification resulted in the least accurate data. In fact, none of the data generated from relative quantification for serine was comparable to that obtained from multipoint calibration. Finally, while single point calibration showed an overall acceptable performance for the majority of the model compounds, we cannot extrapolate the findings to other metabolites within the same analytical run. Analysts are advised to assess accuracy and precision for each metabolite in which single point calibration is the intended quantification mean

Analysis of a Series of Chlorogenic Acid Isomers using Differential Ion Mobility and Tandem Mass Spectrometry

Canada Foundation for InnovationChlorogenic acids are among the most abundant phenolics found in the human diet. Of these, the mono-caffeoylquinic acids are the predominant phenolics found in fruits, such as apples and pears, and products derived from them. In this research, a comprehensive study of the electrospray ionization (ESI) tandem mass spectrometric (MS/MS) dissociation behavior of the three most common mono-caffeoylquinic acids, namely 5-O-caffeoylquinic acid (5-CQA), 3-O-caffeoylquinic acid (3-CQA) and 4-O-caffeoylquinic acid (4-CQA), were determined using both positive and negative ionization. All proposed structures of the observed product ions were confirmed with second-generation MS3 experiments. Similarities and differences between the dissociation pathways in the positive and negative ion modes are discussed, confirming the proposed structures and the established MS/MS fingerprints. MS/MS dissociation was primarily driven via the cleavage of the ester bond linking the quinic acid moiety to the caffeic acid moiety within tested molecules. Despite being structural isomers with the same m/z values and dissociation behaviors, the MS/MS data in the negative ion mode was able to differentiate the three isomers based on ion intensity for the major product ions, observed at m/z 191, 178 and 171. This differentiation was consistent among various MS instruments. In addition, ESI coupled with high-field asymmetric waveform ion mobility spectrometry-mass spectrometry (ESI-FAIMS-MS) was employed for the separation of these compounds for the first time. By combining MS/MS data and differential ion mobility, a method for the separation and identification of mono-caffeoylquinic in apple/pear juice samples was developed with a run time of less than one minute. It is envisaged that this methodology could be used to identify pure juices based on their chlorogenic acid profile (i.e., metabolomics), and could also be used to detect juice-to-juice adulteration (e.g., apple juice addition to pear juice)

Comparative analysis of creatinine and osmolality as urine normalization strategies in targeted metabolomics for the differential diagnosis of asthma and COPD

Peer ReviewedIntroduction Urine is an ideal matrix for metabolomics investigation due to its non-invasive nature of collection and its rich metabolite content. Despite the advancements in mass spectrometry and 1H-NMR platforms in urine metabolomics, the statistical analysis of the generated data is challenged with the need to adjust for the hydration status of the person. Normalization to creatinine or osmolality values are the most adopted strategies, however, each technique has its challenges that can hinder its wide application. Objective Assessment of whether the statistical model established using a targeted urine metabolomics dataset for the differential diagnosis of asthma and chronic obstructive pulmonary disease (COPD) would be improved by normalization to osmolality instead of creatinine. Methods A metabolomics dataset from 51 patient urine samples previously analyzed using two liquid chromatography-tandem mass spectrometry methods was used. The data was normalized to creatinine and osmolality. The statistical analysis was achieved using partial least square discriminant analysis and models of separation were generated and compared. Results Creatinine and osmolality values in asthma and COPD patients were strongly correlated. Using the same metabolites, we found that normalization to osmolality did not significantly change the results. The metabolites of importance in separation remained the same for both methods. The statistical strength of the creatinine model was somewhat better than the osmolality normalized model (R2Q2=0.919, 0.705 vs R2Q2 =0.929, 0.671). Conclusion Our findings suggest that targeted urine metabolomics data can be normalized to creatinine or osmolality with no significant impact on the diagnostic accuracy of the model

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Biosynthesis approach of zinc oxide nanoparticles for aqueous phosphorous removal: physicochemical properties and antibacterial activities

Abstract In this study, phosphorus (PO4 3–-P) is removed from water samples using zinc oxide nanoparticles (ZnO NPs). These nanoparticles are produced easily, quickly, and sustainably using Onion extracts (Allium cepa) at an average crystallite size of 8.13 nm using the Debye–Scherrer equation in the hexagonal wurtzite phase. The characterization and investigation of bio-synthesis ZnO NPs were carried out. With an initial concentration of 250 mg/L of P, the effects of the adsorbent dose, pH, contact time, and temperature were examined. At pH = 3 and T = 300 K, ZnO NPs achieved the optimum sorption capacity of 84 mg/g, which was superior to many other adsorbents. The isothermal study was found to fit the Langmuir model at a monolayer capacity of 89.8 mg/g, and the kinetic study was found to follow the pseudo-second-order model. The adsorption process was verified to be endothermic and spontaneous by thermodynamic characteristics. As a result of their low cost as an adsorbent and their high metal absorption, ZnO NPs were found to be the most promising sorbent in this investigation and have the potential to be used as effective sorbents for the removal of P from aqueous solutions. The antimicrobial activity results showed that ZnO NPs concentration had greater antibacterial activity than conventional Cefotaxime, which was utilized as a positive control in the inhibitory zone. However, no inhibitory zone was visible in the controlled wells that had been supplemented with onion extract and DMSO

Stress Degradation Assessment of Lamotrigine Using a Validated Stability-Indicating HPTLC Method

In this work, a sensitive and stability-indicating HPTLC method for the determination of lamotrigine is presented. According to the International Conference on Harmonization guidelines Q1A, lamotrigine was exposed to a variety of stress conditions; these include heating in acidic, basic and neutral media. Its stability towards oxidative stress, humidity, high temperature and direct sunlight was also examined. Separation of the drug from its forced degradation impurities was achieved using TLC silica gel plates and a mobile phase composed of ethyl acetate: methanol: ammonia. The linear regression analysis of the data obtained for the correlation plots showed good linearity over the concentration range of 10–300 ng/spot. The forced degradation studies showed that lamotrigine is susceptible to degradation under acidic, basic, neutral and oxidative conditions, among which alkaline-induced hydrolysis had the highest degradative potential. Alternatively, the drug was stable under heat, humidity, and daylight stress factors. In order to assess the purity and stability of the drug in tablet formulations, the developed method was applied to the analysis of commercial tablets in brand and generic products. The obtained results showed that the degradation of the drug has not occurred in the marketed formulations that were analyzed by the described methodology

Application of a New Simple Spectrophotometric Method to the Simultaneous Determination of Diclofenac Sodium and Diflunisal in Their Combined Dosage Form

ABSTRACT In this work, a simple and sensitive spectrophotometric method is presented for determination of the non-steriodal anti-inflammatory drugs; diclofenac sodium (DCL) and diflunisal (DIF) in their binary mixture without prior separation. The proposed method is based on the generation of ratio spectra of one compound using the other as the divisor followed by measurement of the peak-to-trough amplitudes between two selected wavelengths in the generated ratio spectra. For the determination of DCL, a standard solution of DIF 5 µg/mL was used as the divisor, and the peak-to-trough amplitudes between 251 and 291 nm were measured and correlated to the corresponding concentrations. Similarly, DCL 7.5 µg/mL was set as the divisor in DIF determination and the peak-to-trough amplitudes at the same wavelengths were recorded. The proposed method was found linear over the concentration ranges 5-50 and 1.5-30 µg/mL for DCL and DIF, respectively. The developed method was validated following the ICH guidelines and successfully applied to the determination of both drugs in various laboratory prepared mixtures. In addition, satisfactory results were obtained from analysis of the commercial pharmaceutical preparation (suppositories) with no significant statistical differences from a reference HPLC method

New simple spectrophotometric method for determination of the binary mixtures (atorvastatin calcium and ezetimibe; candesartan cilexetil and hydrochlorothiazide) in tablets

A new simple spectrophotometric method was developed for the determination of binary mixtures without prior separation. The method is based on the generation of ratio spectra of compound X by using a standard spectrum of compound Y as a divisor. The peak to trough amplitudes between two selected wavelengths in the ratio spectra are proportional to concentration of X without interference from Y. The method was demonstrated by determination of two drug combinations. The first consists of the two antihyperlipidemics: atorvastatin calcium (ATV) and ezetimibe (EZE), and the second comprises the antihypertensives: candesartan cilexetil (CAN) and hydrochlorothiazide (HCT). For mixture 1, ATV was determined using 10 μg/mL EZE as the divisor to generate the ratio spectra, and the peak to trough amplitudes between 231 and 276 nm were plotted against ATV concentration. Similarly, by using 10 μg/mL ATV as divisor, the peak to trough amplitudes between 231 and 276 nm were found proportional to EZE concentration. Calibration curves were linear in the range 2.5â40 μg/mL for both drugs. For mixture 2, divisor concentration was 7.5 μg/mL for both drugs. CAN was determined using its peak to trough amplitudes at 251 and 277 nm, while HCT was estimated using the amplitudes between 251 and 276 nm. The measured amplitudes were linearly correlated to concentration in the ranges 2.5â50 and 1â30 μg/mL for CAN and HCT, respectively. The proposed spectrophotometric method was validated and successfully applied for the assay of both drug combinations in several laboratory-prepared mixtures and commercial tablets. Keywords: Spectrophotometric analysis, Ratio spectra, Atorvastatin, Ezetimibe, Candesartan, Hydrochlorothiazid

Validated HPTLC method for the simultaneous determination of alfuzosin, terazosin, prazosin, doxazosin and finasteride in pharmaceutical formulations

Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases affecting men and it increases in both incidence and prevalence with age. This work presents a simple, sensitive and fast generic high performance thin layer chromatographic (HPTLC) method for the simultaneous determination of five drugs prescribed for the treatment of BPH. These drugs include the α1-adrenergic blockers; alfuzosin hydrochloride (ALF), terazosin hydrochloride (TER), prazosin hydrochloride (PRZ) and doxazosin mesylate (DOX) in addition to the 5α-reductase inhibitor; finasteride (FIN). The cited drugs were separated on TLC-silica plates using a mobile phase composed of methylene chloride:n-hexane:methanol (8.8:0.3:0.9, by volume). Densitometric analysis was carried out at 254 nm for the α-blockers while FIN was measured at 220 nm. The five drugs were detected at Rf values of 0.26, 0.36, 0.45, 0.59 and 0.69 for ALF, TER, PRZ, DOX and FIN, respectively. The developed method was validated according to the International Conference on Harmonization (ICH) guidelines regarding; linearity, ranges, accuracy, precision, selectivity, robustness and limits of detection and quantification. The proposed method showed good linearity (r > 0.9990) in the ranges; 30–350, 30–350, 20–200, 30–350, 200–2000 ng/spot for the cited drugs, respectively. The applicability of the proposed method was verified through the analysis of laboratory-prepared mixtures and percentage recoveries between 98.27% and 101.97% were obtained. Commercial tablets were also analyzed by the developed methodology with no interference detected from the co-formulated excipients. The high sensitivity, simplicity and selectivity of the proposed method suggest its applicability for routine quality-control analysis purposes of any of the titled drugs in their pharmaceutical preparations