The Circulating Biomarkers League: Combining miRNAs with Cell-Free DNAs and Proteins.

The potential of liquid biopsy for the prognosis and diagnosis of diseases is unquestionable. Within the evolving landscape of disease diagnostics and personalized medicine, circulating microRNAs (c-miRNAs) stand out among the biomarkers found in blood circulation and other biological fluids due to their stability, specificity, and non-invasive detection in biofluids. However, the complexity of human diseases and the limitations inherent in single-marker diagnostics highlight the need for a more integrative approach. It has been recently suggested that a multi-analyte approach offers advantages over the single-analyte approach in the prognosis and diagnosis of diseases. In this review, we explore the potential of combining three well-studied classes of biomarkers found in blood circulation and other biofluids-miRNAs, DNAs, and proteins-to enhance the accuracy and efficacy of disease detection and monitoring. Initially, we provide an overview of each biomarker class and discuss their main advantages and disadvantages highlighting the superiority of c-miRNAs over the other classes of biomarkers. Additionally, we discuss the challenges and future directions in integrating these biomarkers into clinical practice, emphasizing the need for standardized protocols and further validation studies. This integrated approach has the potential to revolutionize precision medicine by offering insights into disease mechanisms, facilitating early detection, and guiding personalized therapeutic strategies. The collaborative power of c-miRNAs with other biomarkers represents a promising frontier in the comprehensive understanding and management of complex diseases. Nevertheless, several challenges must be addressed before this approach can be translated into clinical practice.

Role of serum VEGF-A biomarker for early diagnosis of ovarian cancer instead of CA-125.

OBJECTIVE: To assess the significance of serum vascular endothelial growth factor-A (VEGF-A) as a potential biomarker for the diagnosis of ovarian cancer instead of cancer antigen-125 (CA-125). METHODS: The case-control study was conducted at the obstetrics departments of Sir Ganga Ram Hospital and Jinnah Hospital, Lahore, Pakistan, from April to September 2018, and comprised cases of epithelial ovarian tumour and healthy female controls. Serum VEGF-A and CA-125 levels were evaluated using Luminex multi-analyte profiling technology and enzyme immunoassays technique. Age, stage, grading, metastasis and ascites of ovarian cancer patients were investigated and compared with serum VEGF-A and CA-125 levels. Data was analysed using SPSS 20. RESULTS: Of the 89 subjects, 44(49.4%) were cases and 45(49.6%) were controls. Among the cases, 13(29.5%) were benign and 31(70.5%) were malignant. The mean serum VEGF-A values were inversely proportional to the stages of ovarian cancer i.e. stage I, II, III and IV showed 762.2pg/ml, 267.3pg/ml, 233.1pg/ml and 125.5pg/ml VEGF-A levels respectively. A steady increase in the mean serum CA-125 values with the progression of the disease was observed i.e. in stage I, II, III and IV the levels of CA-125 were 146.2U/ml, 268.5U/ml and 477.2U/ml and 844.4U/ml respectively. CONCLUSIONS: The detection of high concentrations of serum VEGF-A level supported its use as one of the diagnostic parameters in the timely investigation of ovarian cancer.

Analytical Validation of HEARTBiT: A Blood-Based Multiplex Gene Expression Profiling Assay for Exclusionary Diagnosis of Acute Cellular Rejection in Heart Transplant Patients.

BACKGROUND: HEARTBiT is a whole blood-based gene profiling assay using the nucleic acid counting NanoString technology for the exclusionary diagnosis of acute cellular rejection in heart transplant patients. The HEARTBiT score measures the risk of acute cellular rejection in the first year following heart transplant, distinguishing patients with stable grafts from those at risk for acute cellular rejection. Here, we provide the analytical performance characteristics of the HEARTBiT assay and the results on pilot clinical validation. METHODS: We used purified RNA collected from PAXgene blood samples to evaluate the characteristics of a 12-gene panel HEARTBiT assay, for its linearity range, quantitative bias, precision, and reproducibility. These parameters were estimated either from serial dilutions of individual samples or from repeated runs on pooled samples. RESULTS: We found that all 12 genes showed linear behavior within the recommended assay input range of 125 ng to 500 ng of purified RNA, with most genes showing 3% or lower quantitative bias and around 5% coefficient of variation. Total variation resulting from unique operators, reagent lots, and runs was less than 0.02 units standard deviation (SD). The performance of the analytically validated assay (AUC = 0.75) was equivalent to what we observed in the signature development dataset. CONCLUSION: The analytical performance of the assay within the specification input range demonstrated reliable quantification of the HEARTBiT score within 0.02 SD units, measured on a 0 to 1 unit scale. This assay may therefore be of high utility in clinical validation of HEARTBiT in future biomarker observational trials.

Validation of an LC-MS/MS-based dilute-and-shoot approach for the quantification of > 500 mycotoxins and other secondary metabolites in food crops: challenges and solutions.

This paper describes the validation of an LC-MS/MS-based method for the quantification of > 500 secondary microbial metabolites. Analytical performance parameters have been determined for seven food matrices using seven individual samples per matrix for spiking. Apparent recoveries ranged from 70 to 120% for 53-83% of all investigated analytes (depending on the matrix). This number increased to 84-94% if the recovery of extraction was considered. The comparison of the fraction of analytes for which the precision criterion of RSD ≤ 20% under repeatability conditions (for 7 replicates derived from different individual samples) and intermediate precision conditions (for 7 technical replicates from one sample), respectively, was met (85-97% vs. 93-94%) highlights the contribution of relative matrix effects to the method uncertainty. Statistical testing of apparent recoveries between pairs of matrices exhibited a significant difference for more than half of the analytes, while recoveries of the extraction showed a much better agreement. Apparent recoveries and matrix effects were found to be constant over 2-3 orders of magnitude of analyte concentrations in figs and maize, whereas the LOQs differed less than by a factor of 2 for 90% of the investigated compounds. Based on these findings, this paper discusses the applicability and practicability of current guidelines for multi-analyte method validation. Investigation of (apparent) recoveries near the LOQ seems to be insufficiently relevant to justify the enormous time-effort for manual inspection of the peaks of hundreds of analytes. Instead, more emphasis should be put on the investigation of relative matrix effects in the validation procedure. Graphical abstract.

Development and validation of a multi-analyte GC-MS method for the determination of 84 substances from plastic food contact materials.

Chemical substances shall not migrate from food contact materials (FCM) at levels that are potentially harmful for the consumers. Each of the current analytical methods applied to verify the migration of substances from FCM covers only one or few substances. There is a very limited number of publications on the development of analytical methods allowing the simultaneous determination of several classes of FCM substances, and almost none of them reported methods entirely dedicated to the ones in the positive list of Commission Regulation (EU) No. 10/2011 for plastic FCMs. Therefore, a simple, sensitive and reliable multi-analyte method was developed for the analysis of FCM substances in food simulants. It employs an optimised liquid-liquid extraction with dichloromethane as extraction solvent in the presence of 10% m/v NaCl, followed by quantitative analysis with gas chromatography coupled to mass spectrometry (GC-MS). A combination of total ion chromatograms (TICs) and extracted ion chromatograms (EICs) was used. The optimisation and validation of the method have been carried out according to current international guidelines. Adequate sensitivity was demonstrated in the selected concentration ranges for most of the analytes, with limits of quantification (LOQs) at least three times lower than the legislative limit, when existing. The results showed that the method is sufficiently accurate for the majority of substances, with recoveries between 70 and 115% and relative standard deviations (RSDs) smaller than 20% at three concentration levels. The method was applied to the analysis of some FCM multilayers. The method allows, for the first time, the simultaneous quantification of 84 FCM substances in two of the official food simulants (A and C) at levels of a few ng g-1. Graphical abstract.

Derivatization-free determination of short-chain volatile amines in human plasma and urine by headspace gas chromatography-mass spectrometry.

BACKGROUND: Short-chain volatile amines (SCVA) are an interesting compound class playing crucial roles in physiological and toxicological human settings. Dimethylamine (DMA), trimethylamine (TMA), diethylamine (DEA), and triethylamine (TEA) were investigated in detail. METHODS: Headspace gas chromatography coupled to mass spectrometry (HS-GC-MS) was used for the simultaneous qualitative and quantitative determination of four SCVA in different human body fluids. Four hundred microliters of Li-heparin plasma and urine were analyzed after liberation of volatile amines under heated conditions in an aqueous alkaline and saline environment. Target analytes were separated on a volatile amine column and detected on a Thermo DSQ II mass spectrometer scheduled in single ion monitoring mode. RESULTS: Chromatographic separation of selected SCVA was done within 7.5 minutes. The method was developed and validated with respect to accuracy, precision, recovery and stability. Accuracy and precision criteria were below 12% for all target analytes at low and high levels. The selected extraction procedure provided recoveries of more than 92% from both matrices for TMA, DEA and TEA. The recovery of DMA from Li-heparin plasma was lower but still in the acceptable range (>75%). The newly validated method was successfully applied to plasma and urine samples from healthy volunteers. Detected concentrations of endogenous metabolites DMA and TMA are comparable to already known reference ranges. CONCLUSION: Herein, we describe the successful development and validation of a reliable and broadly applicable HS-GC-MS procedure for the simultaneous and quantitative determination of SCVA in human plasma and urine without relying on derivatization chemistry.

Antibody-Electroactive Probe Conjugates Based Electrochemical Immunosensors.

Suitable immobilization of a biorecognition element, such as an antigen or antibody, on a transducer surface is essential for development of sensitive and analytically reliable immunosensors. In this review, we report on (1) methods of antibody prefunctionalization using electroactive probes, (2) methods for immobilization of such conjugates on the surfaces of electrodes in electrochemical immunosensor construction and (3) the use of antibody-electroactive probe conjugates as bioreceptors and sensor signal generators. We focus on different strategies of antibody functionalization using the redox active probes ferrocene (Fc), anthraquinone (AQ), thionine (Thi), cobalt(III) bipyridine (Co(bpy)33+), Ru(bpy)32+ and horseradish peroxidase (HRP). In addition, new possibilities for antibody functionalization based on bioconjugation techniques are presented. We discuss strategies of specific, quantitative antigen detection based on (i) a sandwich format and (ii) a direct signal generation scheme. Further, the integration of different nanomaterials in the construction of these immunosensors is presented. Lastly, we report the use of a redox probe strategy in multiplexed analyte detection.

A quartz crystal microbalance modified with antibody-coated silver nanoparticles acting as mass signal amplifiers for real-time monitoring of three latent tuberculosis infection biomarkers.

A strategy is described for continuous monitoring of multiple latent tuberculosis infection (LTBI) biomarkers, specifically of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2). Silver nanoparticles acting as mass signal amplifiers were linked to respective antibodies to form mass nanoprobes for increasing the mass loaded on the surface of the quartz crystal microbalance (QCM). This results in enhanced sensitivity. The mass nanoprobes can be oxidatively dissolved by hydrogen peroxide that avoided the steric hindrance caused by the scale effect of mass nanoprobes. This offers the option of signal recovery monitoring. By using this method, IFN-γ, TNF-α and IL-2 can be monitored serially. The frequency shifts caused by TNF-α, IFN-γ and IL-2, respectively, are reversible. Hence, the biomarkers can be continuously quantified. Compared to multichannel QCM sensing, the new method avoids acoustic interference and has a simplified instrumental setup. The assay is simple, accurate, sensitive, and inexpensive. Graphical abstract Silver nanoparticles as the mass signal amplifiers were linked with the antibodies to form mass nanoprobes for enhancing the monitoring sensitivity. With the introduction of H2O2 to dissolve the mass nanoprobes attached on sensing interface, a signal recovery QCM strategy is established for real-time and continuous monitoring of three LTBI biomarkers.

Simultaneous determination of two phosphorylated p53 proteins in SCC-7 cells by an ICP-MS immunoassay using apoferritin-templated europium(III) and lutetium(III) phosphate nanoparticles as labels.

Phosphorylated p53 proteins are biomarkers with clinical utility for early diagnosis of cancer, but difficult to quantify. An inductively coupled plasma mass spectrometry (ICP-MS) based immunoassay is described here that uses uniform lanthanide nanoparticles (NPs) as elemental tags for the simultaneous determination of two phosphorylated p53 proteins. Apoferritin templated europium (Eu) phosphate (AFEP) NPs and apoferritin templated lutetium (Lu) phosphate (AFLP) NPs with 8 nm in diameter were used to label two phosphorylated p53 proteins at serine 15 and serine 392 sites (p-p5315 and p-p53392), respectively. The assay has a sandwich format, and p-p5315 and p-p53392 were first captured and then recognized by AFEP and AFLP NPs labelled antibodies, respectively. The Eu and Lu were then released from the immune complexes under acidic condition for ICP-MS measurement. The limits of detection for p-p5315 and p-p53392 are 200 and 20 pg·mL-1, with linear ranges of 0.5-20 and 0.05-20 ng·mL-1, respectively. The method was further applied to study the response of p-p5315 and p-p53392 in SCC-7 cells exposed to the natural carcinogen arsenite. A significant up-regulation of p-p5315 and p-p53392 can be observed when cells were exposed to arsenite at 5 µmol·L-1 level for 24 h. Graphical abstract Schematic presentation of the ICP-MS immunoassay using apoferritin templated europium (III) and lutetium (III) phosphate nanoparticles as labels for the simultaneous determination of two phosphorylated p53 proteins. Europium (Eu) phosphate nanoparticles (blue) and lutetium (Lu) phosphate nanoparticles (pink) were synthesized in the size-restricted cavity of apoferritin. They were further coupled with antibodies to prepare Eu and Lu labelled probes for p-p5315 (blue) and p-p53392 (pink), respectively. After formation of a a sandwich, the labelled Eu and Lu were dissociated in acid and then introduced to ICP-MS for the simultaneous determination of two phosphorylated p53 proteins p-p5315 (blue) and p-p53392 (pink).

A Minimally Invasive Microsensor Specially Designed for Simultaneous Dissolved Oxygen and pH Biofilm Profiling.

A novel sensing device for simultaneous dissolved oxygen (DO) and pH monitoring specially designed for biofilm profiling is presented in this work. This device enabled the recording of instantaneous DO and pH dynamic profiles within biofilms, improving the tools available for the study and the characterization of biological systems. The microsensor consisted of two parallel arrays of microelectrodes. Microelectrodes used for DO sensing were bare gold electrodes, while microelectrodes used for pH sensing were platinum-based electrodes modified using electrodeposited iridium oxide. The device was fabricated with a polyimide (Kapton®) film of 127 µm as a substrate for minimizing the damage caused on the biofilm structure during its insertion. The electrodes were covered with a Nafion® layer to increase sensor stability and repeatability and to avoid electrode surface fouling. DO microelectrodes showed a linear response in the range 0-8 mg L-1, a detection limit of 0.05 mg L-1, and a sensitivity of 2.06 nA L mg-1. pH electrodes showed a linear super-Nernstian response (74.2 ± 0.7 mV/pH unit) in a wide pH range (pH 4-9). The multi-analyte sensor array was validated in a flat plate bioreactor where simultaneous and instantaneous pH and DO profiles within a sulfide oxidizing biofilm were recorded. The electrodes spatial resolution, the monitoring sensitivity, and the minimally invasive features exhibited by the proposed microsensor improved biofilm monitoring performance, enabling the quantification of mass transfer resistances and the assessment of biological activity.

A LAPS-Based Differential Sensor for Parallelized Metabolism Monitoring of Various Bacteria.

Monitoring the cellular metabolism of bacteria in (bio)fermentation processes is crucial to control and steer them, and to prevent undesired disturbances linked to metabolically inactive microorganisms. In this context, cell-based biosensors can play an important role to improve the quality and increase the yield of such processes. This work describes the simultaneous analysis of the metabolic behavior of three different types of bacteria by means of a differential light-addressable potentiometric sensor (LAPS) set-up. The study includes Lactobacillus brevis, Corynebacterium glutamicum, and Escherichia coli, which are often applied in fermentation processes in bioreactors. Differential measurements were carried out to compensate undesirable influences such as sensor signal drift, and pH value variation during the measurements. Furthermore, calibration curves of the cellular metabolism were established as a function of the glucose concentration or cell number variation with all three model microorganisms. In this context, simultaneous (bio)sensing with the multi-organism LAPS-based set-up can open new possibilities for a cost-effective, rapid detection of the extracellular acidification of bacteria on a single sensor chip. It can be applied to evaluate the metabolic response of bacteria populations in a (bio)fermentation process, for instance, in the biogas fermentation process.

Development of a simple HPLC-DAD multi-analyte procedure and its application in cases evaluated by the Poison Control Center of São Paulo, Brazil.

This work describes a simple approach to overcome challenges in emergency toxicological analysis, using liquid-liquid extraction and high-performance liquid chromatography coupled with a diode-array detector (HPLC-DAD). A rapid procedure has been developed, for the extraction and detection of 19 analytes from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants and drugs of abuse. These substances are relevant in the context of emergency toxicology in Brazil. The method has been validated according to international guidelines by establishing parameters such as lower limit of quantification, sensitivity, linearity, accuracy and precision. The intra and inter-day precision values, at the lowest concentration levels, have always been less than 20% considering its relative standard deviation. As for accuracy values, these have also been satisfactory (above 81.3%). This method was successfully applied in 201 blood samples from patients with suspected poisoning of the Poison Control Center of São Paulo (PCC-SP), Brazil. Finally, the developed method has shown to be relevant for emergency toxicology due to its high sensitivity and it could be also very useful in both fields of clinical and forensic toxicology.

Application of a Portable Multi-Analyte Biosensor for Organic Acid Determination in Silage.

Multi-analyte biosensors may offer the opportunity to perform cost-effective and rapid analysis with reduced sample volume, as compared to electrochemical biosensing of each analyte individually. This work describes the development of an enzyme-based biosensor system for multi-parametric determination of four different organic acids. The biosensor array comprises five working electrodes for simultaneous sensing of ethanol, formate, d-lactate, and l-lactate, and an integrated counter electrode. Storage stability of the biosensor was evaluated under different conditions (stored at +4 °C in buffer solution and dry at −21 °C, +4 °C, and room temperature) over a period of 140 days. After repeated and regular application, the individual sensing electrodes exhibited the best stability when stored at −21 °C. Furthermore, measurements in silage samples (maize and sugarcane silage) were conducted with the portable biosensor system. Comparison with a conventional photometric technique demonstrated successful employment for rapid monitoring of complex media.

Toxicological screening of human plasma by on-line SPE-HPLC-DAD: identification and quantification of acidic and neutral drugs.

A multi-analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on-line solid-phase extraction (SPE)-HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro-neutral, one strong anion-exchange, one weak cation-exchange) for on-line extraction, five HPLC-columns [one C18 (GeminiNX), two phenyl-hexyl (Gemini C6 -Phenyl, Kinetex Phenyl-Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre-treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion-exchanger SPE cartridge (StrataX-A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C6 -Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile-water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter-/intra-day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from -14 to 10%. A computer-assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi-drug intoxications are presented.

Development of a bead-based aptamer/antibody detection system for C-reactive protein.

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10mg/L in diluted serum with acceptable recoveries (extrapolated values of 70-130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer-target systems could increase the number of analytes measurable using xMAP-type assays.

Toxicological screening of human plasma by on-line SPE-HPLC-DAD: identification and quantification of basic drugs and metabolites.

An automated multi-analyte screening method for the identification and quantification of 92 drugs and metabolites based on on-line solid-phase extraction-high-performance liquid chromatography-diode array detection technique was developed and successfully validated. In addition, a database with 870 entries including UV-spectra, relative/retention times and response factors of toxicologically relevant compounds was created. Plasma samples (0.2 mL) were treated with methanol, diluted with buffer and on-line extracted (Strata X, 20 ×2 mm, 25 µm) at pH 9. Analytical separation was carried out on a Gemini NX column (150 ×4.6 mm, 3 µm) using gradient elution with acetonitrile-water (90:10,v/v) and 0.05 m potassium dihydrogen phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients ≥0.9950 were obtained for 78 analytes. As an additional benefit, the newly developed method allows the quantification of 42 analytes (e.g. antidepressants, neuroleptics and anticonvulsants) in a concentration range suitable for therapeutic drug monitoring. Limits of quantitation ranged from 0.02 mg/L (chlordiazepoxide) to 3.4 mg/L (mexiletine). Inter- and intra-day precisions of quality control samples (low/high) were better than 15% (zolpidem) and accuracy (bias) ranged from -11% (opipramol, venlafaxine) to 11% (venlafaxine, trazodone). Tests for carry-over and sample stability under different storage conditions were also performed and stability was adequate. Four cases of poisoning analysis are presented.

Host cell protein testing by ELISAs and the use of orthogonal methods.

Host cell proteins (HCPs) are among the process-related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process-related impurities are needed to support process development, process validation, and control system testing. A multi-analyte enzyme-linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti-HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non-immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution-dependent non-linearity with multi-product HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co-purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low-level HCPs is also outlined, with consideration of clinical information.

A multi-analyte assay of opioids in API and drug products using HPLC and HRMS.

Surveillance testing is an essential component to ensuring safe, effective, and high-quality drug products are available in the commercially marketed US supply chain. Surveillance allows the agency to assess product quality and monitor for potential adulteration of drug products being used by consumers. Opioid drug products can be adulterated to enhance the effect of the intended active ingredient. Numerous accounts have been reported where fentanyl has been used as an adulterant in illicit street drugs such as heroin, cocaine, or methamphetamine. To efficiently surveil the legitimate opioid supply chain, an analytical method with the ability to simultaneously detect, identify and quantify opioid molecules is desired. In this study, a multi-opioid protocol (MOP) using liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) technology was developed and validated for the detection and quantification of 27 opioid drugs. The MOP analytical procedure was applied to the analysis of drug substance and finished dosage forms. MOP was used to identify and quantify active pharmaceutical ingredients (API) listed on the label claim, and in the case of suspected economically motivated adulteration could identify and quantify undeclared opioid APIs. The analytical method analysis time was 16 minutes and the LOD and LOQ in full MS mode were (average) 0.3 and 0.8 ng/mL, respectively. The validation criteria parameters were satisfactory based on international guidelines (ICH). The MOP was successfully applied to the analysis of over 160 drug substances and finished products. For all samples tested in the study, their identities were confirmed, and assays met specifications. Overall, there was no evidence of illegal substitution or adulteration in any of the ingredients and products tested from the legitimate commercial marketed US supply chain.

Accuracy and Feasibility of a Novel Glucose/Lactate Continuous Multi-Analyte Sensing Platform in Humans.

BACKGROUND AND AIM: Continuous glucose monitoring systems (CGMs) have been commercially available since 1999. However, automated insulin delivery systems may benefit from real-time inputs in addition to glucose. Continuous multi-analyte sensing platforms will meet this area of potential growth without increasing the burden of additional devices. We aimed to generate pilot data regarding the safety and function of a first-in-human, single-probe glucose/lactate multi-analyte continuous sensor. METHODS: The investigational glucose/lactate continuous multi-analyte sensor (PercuSense Inc, Valencia, California) was inserted to the upper arms of 16 adults with diabetes, and data were available for analysis from 11 of these participants (seven female; mean [SD] = age 43 years [16]; body mass index [BMI] = 27 kg/m2 [5]). A commercially available Guardian 3 CGM (Medtronic, Northridge, California) was also inserted into the abdomen for comparison. All participants underwent a meal-test followed by an exercise challenge on day 1 and day 4 of wear. Performance was benchmarked against venous blood YSI glucose and lactate values. RESULTS: The investigational glucose sensor had an overall mean absolute relative difference (MARD) of 14.5% (median = 11.2%) which improved on day 4 compared with day 1 (13.9% vs 15.2%). The Guardian 3 CGM had an overall MARD of 13.9% (median = 9.4%). The lactate sensor readings within 20/20% and 40/40% of YSI values were 59.7% and 83.1%, respectively. CONCLUSIONS: Our initial data support safety and functionality of a novel glucose/lactate continuous multi-analyte sensor. Further sensor refinement will improve run-in performance and accuracy.