Acetaminophen Assay Research Articles

Agreement Between a Colorimetric Assay and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry for Quantifying Paracetamol Plasma Concentrations.

Special populations, like geriatric patients, experience altered paracetamol pharmacokinetics (PK), complicating pain management. More PK research is essential to optimize paracetamol (acetaminophen) dosing. Yet, the reference method ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is not readily available. Therefore, we aimed to evaluate the agreement between UPLC-MS/MS and the more accessible colorimetric Roche acetaminophen (ACETA) assay in quantifying paracetamol plasma concentrations, to facilitate PK studies and therapeutic drug monitoring for pain management. Patient data and plasma samples were obtained from a prospective study including geriatric patients admitted to the geriatric wards. ACETA and UPLC-MS/MS assays were performed in two separate laboratories. Bland-Altman plot and Passing-Bablok regression were used to assess agreement. Accuracy was evaluated using the McNemar test for a threshold value of 10mg/L. Population PK modeling was employed to bridge PK data obtained from both methods (NONMEM 7.5). A total of 242 plasma sample pairs were available from 40 geriatric patients (age range, 80-95years). Paracetamol plasma concentrations from ACETA (median 9.8 [interquartile range 6.1-14.4] mg/L) and UPLC-MS/MS (9.5 [6.2-14.8] mg/L) did not differ significantly (P > 0.05). No significant proportional nor additive bias was observed between both assay methods. The classification accuracy (at threshold 10mg/L) was 85% (P = 0.414). The conversion factor between ACETA and UPLC-MS/MS was estimated at 1.06 (relative standard error 5%), yet with a 13.4% (relative standard error 23%) interindividual variability. ACETA assay showed no systematic bias in comparison with the UPLC-MS/MS assay in determining paracetamol exposure in geriatric blood samples despite the imprecision.

A-130 Analytical Performance Evaluation of Assays Measuring Acetaminophen and Ethanol on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica CH Ethyl Alcohol (ETOH) and Acetaminophen (Acet) assays on the Atellica CI 1900 Analyzer. Methods The Atellica CI 1900 ETOH and Acet assays use the same reagents and calibrators as the comparable Atellica CH assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3. Native and contrived human serum samples were used. One aliquot of each sample pool was tested in duplicate in two runs per day >2 h apart on each analyzer for >20 days. MC studies were performed according to CLSI EP09-A3. Individual human serum samples were analyzed using the Atellica CH assays on both the Atellica CH and Atellica CI 1900 Analyzers. Precision and MC were evaluated with three reagent lots on one system. Results Representative precision and MC results observed for each assay are listed in the table. Over the two assays tested, repeatability and within-lab %CVs were <2.5% and <4.0%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1 for the ETOH and Acet assays. Conclusion Evaluation of the Atellica CH ETOH and Acet assays using the Atellica CI 1900 Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica CH Analyzer. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

Evaluation of analytical performances of a serum acetaminophen assay designed for round-the-clock use

Acetaminophen (paracetamol) voluntary (or not) intoxications are a major cause of life-threatening liver damage. Most of available assays on the market show an upper range measurement level that is close to thresholds considered as toxic levels. Therefore, high concentration samples must be further diluted in to accurately measure paracetamol levels. This study aims at assessing the performances of a Cambridge life sciences (CLS) test for acetaminophen determination in human serum, which solves this issue. Indeed, the particularities of this test consist in (i) an extended announced upper limit of quantitation, and (ii) in the use of a genetically unique enzyme (Aryl acylamidase). This evaluation encompasses comparison of results with those obtained using the acetaminophen Thermofisher test currently used in the University Hospital of Lille, and here after referred to as “reference method”. Analytical performance was evaluated on a Beckman Coulter AU480 chemistry analyzer. Within-run precision was determined by assaying 3 serum samples at 3 concentration levels (low, medium, high) in 10 replicates and between-run precision in running 5 samples on 6 days, twice a day. Linearity was assessed against a set of standards covering the entire claimed linearity range. Fifty-two fresh serum samples form patients were prospectively assessed for correlation to the reference method. The within-run and between-run precision (%CV) was respectively < 1% and < 3%. The assay showed good linearity with very low CV < 2% at concentrations up to 859 mg/L, consistent with the analytical range announced by the manufacturer. The regression equation against the reference method was Y = 1.133 x + 0.145, r2 = 0.99988. However, the lower limit of quantification (LLOQ) appears to be slightly higher than the manufacturer's stated (3 mg/L). Comparison to reference method showed that values given by the CLS assay were overestimated especially in the low range of concentration values (< 50 mg/L). The CLS acetaminophen assay has proven to have good analytical performance. Non-correlation to reference method does not affect the ability of the test to detect and to guide the management of paracetamol poisoning. The major advantage of CLS acetaminophen assay is the broad linearity range covering high acetaminophen concentrations. The use of this assay eliminates the need for dilution of highly positive samples and is particularly designed for round-the-clock use.

Simple colorimetric paper assay of acetaminophen, ascorbic acid and phenylephrine in pharmaceutical samples

Simple colorimetric paper assay of acetaminophen, ascorbic acid and phenylephrine in pharmaceutical samples

Routine Laboratory Screening for Acetaminophen and Salicylate Ingestion in Preadmission Psychiatric Patients Is Unnecessary

Screening preadmission psychiatric patients for acetaminophen or salicylate overdose is unnecessary in the absence of specific clinical concern for medication ingestion. This was a multicenter retrospective cohort study of 3 Veteran's Administration emergency departments that medically evaluate patients prior to psychiatric admission. During the 10-year study period, these departments followed screening protocols that required the measurement of acetaminophen and salicylate levels on every patient prior to psychiatric admission. We examined all the acetaminophen and salicylate assays performed to see if any that were sent for screening led to a diagnosis of overdose and/or the administration of antidotal therapy. A total of 33,439 combined acetaminophen and salicylate assays were sent on 10,482 unique patients over approximately 17,000 patient encounters. An estimated 29,000 assays were sent for screening purposes only-87% (95% confidence interval [CI] 85% to 89%) of salicylate assays and 85% (95% CI 83% to 87%) of acetaminophen assays. We identified 43 patients with elevated acetaminophen levels and 11 with elevated salicylate levels. Among these patients, only 6 in total had their levels drawn for screening purposes only, with no history of suspected ingestion; in all but 1 patient, the levels were only slightly above the reference range. None of the patients with elevated levels identified by screening had clinical toxicity or received antidotal therapy. Over a 10-year period, 3 Veteran's Administration emergency departments performed psychiatric preadmission screening protocols with acetaminophen and salicylate assays approximately 17,000 times without diagnosing a single case of toxicity. Our results suggest that this practice is unnecessary and wasteful.

A Computational Approach to Identify Interfering Medications on Urine Drug Screening Assays without Data from Confirmatory Testing.

Urine drug screening (UDS) assays can rapidly and sensitively detect drugs of abuse but can also produce spurious results due to interfering substances. We previously developed an approach to identify interfering medications using electronic health record (EHR) data, but the approach was limited to UDS assays for which presumptive positives were confirmed using more specific methods. Here we adapted the approach to search for medications that cause false positives on UDS assays lacking confirmation data. From our institution’s EHR data, we used our previous dataset of 698,651 UDS and confirmation results. We also collected 211,108 UDS results for acetaminophen, ethanol and salicylates. Both datasets included individuals’ prior medication exposures. We hypothesized that the odds of a presumptive positive would increase following exposure to an interfering medication independently of exposure to the assay’s target drug(s). For a given assay–medication pair, we quantified potential interference as an odds ratio from logistic regression. We evaluated interference of selected compounds in spiking experiments. Compared to the approach requiring confirmation data, our adapted approach showed only modestly diminished ability to detect interfering medications. Applying our approach to the new data, we discovered and validated multiple compounds that can cause presumptive positives on the UDS assay for acetaminophen. Our approach can reveal interfering medications using EHR data from institutions at which UDS results are not routinely confirmed.

Disposable Nafion-Coated Single-Walled Carbon Nanotube Test Strip for Electrochemical Quantitative Determination of Acetaminophen in a Finger-Prick Whole Blood Sample

A disposable electrochemical test strip for the quantitative point-of-care (POC) determination of acetaminophen (paracetamol) in plasma and finger-prick whole blood was fabricated. The industrially scalable dry transfer process of single-walled carbon nanotubes (SWCNTs) and screen printing of silver were combined to produce integrated electrochemical test strips. Nafion coating stabilized the potential of the Ag reference electrode and enabled the selective detection in spiked plasma as well as in whole blood samples. The test strips were able to detect acetaminophen in small 40 μL samples with a detection limit of 0.8 μM and a wide linear range from 1 μM to 2 mM, well within the required clinical range. After a simple 1:1 dilution of plasma and whole blood, a quantitative detection with good recoveries of 79% in plasma and 74% in whole blood was achieved. These results strongly indicate that these electrodes can be used directly to determine the unbound acetaminophen fraction without the need for any additional steps. The developed test strip shows promise as a rapid and simple POC quantitative acetaminophen assay.

Development and validation of assay and impurities methods for acetaminophen and diphenhydramine tablets

AbstractThe United States Pharmacopeia compendium does not currently contain a documentary standard for acetaminophen and diphenhydramine hydrochloride tablets to determine quality. To address this gap, high‐performance liquid chromatography methods for assay and impurities were developed and validated. Marketed products contain different amounts of drug substances per unit (325–500 mg of acetaminophen and 12.5–25 mg of diphenhydramine), so two methods specific to each substance were required. Methods were designed for high throughput by limiting analysis time, and complexity was reduced by utilizing similar materials and instrumental configurations across methods. For each method, specificity, linearity, accuracy, and precision were demonstrated for the assay and impurity determination. Both methods were shown to be fit‐for‐purpose and may be considered for adoption as United States Pharmacopeia compendial standards.

IMPROVISED AND VALIDATED OF TWO SPECTROPHOTOMETRY METHOD FOR PARALEL ASSAY OF GUAIPHENESIN, ACETAMINOPHEN AND CTM COMBINE IN SYRUP

IMPROVISED AND VALIDATED OF TWO SPECTROPHOTOMETRY METHOD FOR PARALEL ASSAY OF GUAIPHENESIN, ACETAMINOPHEN AND CTM COMBINE IN SYRUP

Sensitive Detection of Acetaminophen Based on N, P-Co-Doped Carbon Microspheres Modified Electrode

Heteroatom doped carbon materials have attracted intense attention in constructing electrochemical sensors because of their outstanding electrocatalytic performance. Up to now, most of the researches were focused on the electrochemical application of single heteroatom doped carbon microspheres. In this work, N, P-co-doped carbon microspheres (NP-CMs) modified electrode was developed as highly sensitive electrochemical sensor for acetaminophen. NP-CMs were acquired by the calcination of products from the one-pot reaction of dopamine and 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO). Experimental results revealed that NP-CMs showed better electrocatalytic performance compared with N-doped carbon microspheres. Under the optimal conditions, the response of NP-CMs/GCE showed a linear relationship with acetaminophen concentration from 0.07 μM to 30 μM, the linear equation was Y = 0.01356X+0.7417 and the detection limit was 40 nM (S/N = 3). The recoveries of the NP-CMs modified GCE for the assay of acetaminophen in human serum samples were from 94.0% to 106.0%. It was believed that N, P-co-doped carbon microspheres have a great potential in the design of novel pharmaceutical analysis methods.

Effect of hemolysis, icterus, and lipemia on three acetaminophen assays: Potential medical consequences of false positive results

BackgroundWe evaluated the effect of hemolysis, icterus and lipemia on 3 acetaminophen assays: namely the Syva® EMIT®, the Microgenics DRI® assay, and the Roche assay on a Roche Cobas® c501 or an Integra 800 analyzer. MethodsDiscarded acetaminophen – free serum samples (blank pool) and patient serum with acetaminophen overdose were used to prepare samples. Three levels of acetaminophen (5, 10, and 30 μg/ml) were evaluated for interference: hemolysis (H index range: 0–1000), icterus (I index range: 0–40), and lipemia (L index range: 0–1000). ResultsMeasurements showed that the EMIT® assay was not significantly affected by hemolysis or icterus at all 3concentrations evaluated, but was negatively affected by lipemia at all three levels at 1000 mg/dl intralipids. The DRI® assay was similarly affected by hemolysis and icterus, but lipemia (at 1000 mg/dl intralipids) only affected the 5 μg/ml level. The Roche acetaminophen assay was significantly affected by hemolysis at all three concentrations. It was significantly affected by icterus at 20 mg/dl bilirubin and > 5 μg/ml and at icterus levels of 30 and 40 mg/dl bilirubin at 10 and 30 μg/ml acetaminophen concentrations, respectively. However, the Roche assay was least affected by lipemia. ConclusionHemolysis and icterus had insignificant interference on the Syva EMIT® and the DRI® assays for the analysis of acetaminophen, but significant interference effect on the Roche assay. On the other hand lipemia interfered less markedly with the Roche assay. The effect of hemolysis, icterus and lipemia should always be considered. Cautions are warranted when interpreting results for the potential false positive results in the presence of hemolysis and icterus at the concentrations evaluated in this study.

Evaluation of a single-dose PIGRET assay for acetaminophen in rats compared with the RBC Pig-a assay

As a part of a collaborative study of the Pig-a assay by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society, a genotoxicity study on acetaminophen (APAP) was performed using the red blood cell (RBC) Pig-a and PIGRET assays. The dose levels were set at 0 (vehicle, 0.5% methylcellulose solution), 500, 1000, and 2000mg/kg, and APAP was administered once by oral gavage to male Sprague Dawley rats. For the positive control group, N-nitroso-N-ethylurea (ENU, 40mg/kg) was administered in the same way. The RBC Pig-a and PIGRET assays were performed using peripheral blood collected at pre-dosing and 1, 2 and 4 weeks after dosing. In both the RBC Pig-a and PIGRET assays, there were no changes in the Pig-a gene mutant frequency (MF) by the APAP treatment at any time point. The Pig-a MFs as measured by the RBC Pig-a assay for the ENU-treated group increased in a time-dependent manner with the maximum value at week 4; however, those using the PIGRET assay reached comparable values at week 1. Based on the above results, APAP was determined to have no mutagenicity under the conditions of this study, and the PIGRET assay could detect mutagenicity of ENU much earlier than the RBC Pig-a assay.

Modification of carbon paste electrode with Fe(III)-clinoptilolite nano-particles for simultaneous voltammetric determination of acetaminophen and ascorbic acid.

A novel carbon paste electrode (CPE) modified with Fe(III)-exchanged clinoptilolite nano-particles (Fe(III)-NClino/CPE) was constructed and used for simultaneous voltammetric (CV, SqW and chronoamperometry) determination of ascorbic acid and acetaminophen. Raw and modified zeolites were characterized by X-ray diffraction (XRD) and transmission electron microscope (TEM). The square wave peak current was linearly increased in the concentration ranges of 1.0 × 10(-9)-1.0 × 10(-2) mol L(-1) for ascorbic acid and 1.0 × 10(-10-)1.0 × 10(-2) mol L(-1) for acetaminophen with detection limits of 1.8 × 10(-9) mol L(-1) and 9.9 × 10(-10) mol L(-1), respectively. The detection limits of 2.4 × 10(-10) mol L(-1) and 2.5 × 10(-11) mol L(-1) were also obtained for AA and AC in chronoamperometric measurements, respectively. The diffusion coefficients of 7.5 × 10(-5) cm(2) s(-1) and 2.4 × 10(-5) cm(2) s(-1) were respectively calculated for the oxidation of AC and AA by chronoamperometry. The proposed electrode exhibited high sensitivity and good stability, and would be valuable for the clinical assay of ascorbic acid and acetaminophen.

Bi-variate approach to negative interference of bilirubin towards an acetaminophen assay

BackgroundIt is well known that enzymatic assays for acetaminophen are positively interfered by bilirubin. The effect on acetaminophen not only depends on the concentration of bilirubin but also on that of acetaminophen. We demonstrated a negative interference instead of a positive one in a commonly used routine analyzer and investigated the recovery of acetaminophen in an enzymatic assay by a bi-variate regression. MethodsCommercially available blank serum specimens were spiked with acetaminophen and bilirubin at various concentrations, and were analyzed in the Beckman Coulter AU5822 analyzer. The specimens were run in duplicates. The results were then analyzed by least-square analysis and was built into a bi-variate quadratic model. ResultsThe recovery of acetaminophen in this experiment ranged from 38.9% to 100% throughout a range of 23μmol/L to 2052μmol/L (for acetaminophen) and 19μmol/L to 570μmol/L (for bilirubin). A contour map, as well as a bi-variate equation was established, describing the relationship between acetaminophen recovery, acetaminophen concentration, and bilirubin concentration. ConclusionIt was shown that the degree of bilirubin interference in a commercially available acetaminophen assay is dependent on both bilirubin and acetaminophen concentrations. There was a decrease in the apparent acetaminophen concentration by an average of 30% at a bilirubin concentration of 420μmol/L in the Beckman Coulter AU5822 analyzer. The complex relationship can be modeled by mathematical means. This allows the laboratory staff to estimate the recovery of acetaminophen when bilirubin level is concurrently measured.

Magnetic beads as an extraction medium for simultaneous quantification of acetaminophen and structurally related compounds in human serum.

This paper describes a sample preparation method that complements a previously published liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for acetaminophen and eight structurally-related compounds in human serum (C. Bylda, R. Thiele, U. Kobold, D.A. Volmer. Drug Test. Anal. 2014, 6, 451). The analytes (acetaminophen [APAP] + metabolites acetaminophen-glucuronide [APG], -cysteine [APC], -mercapturate [APM] and -cysteine [APC], structurally similar analogues phenacetin and p-phenetidine, as well as tricyclic antidepressants imipramine and amitryptiline) were extracted from serum using magnetized hyper-crosslinked polystyrene particles. The sample preparation protocol was developed by means of a design of experiments (DoE) statistical approach. Using three representative compounds from the analyte panel with different polarities (high, medium, and low), two screening designs were used to identify factors that exhibited significant impact on recovery of the analytes. These parameters were then optimized to permit extraction of the complete target panel exhibiting a broad range of chemical polarities. Liquid chromatographic separations were achieved by gradient elution using a pentafluorphenyl column with subsequent detection by electrospray ionization-triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode. The method was linear over the range 0.1-100 µg/mL for APAP, APG, p-phenetidine and phenacetin, 0.03-50 µg/mL for APS, and 0.01-10 µg/mL for APM, APC, imipramine and amitriptyline, with R(2) > 0.99. The assay exhibited good precision with CVs ranging from 2 to 9% for all analytes; the accuracy was assessed by comparing two LC-MS/MS methods using a set of 68 patient samples.

Differences in bilirubin and biliverdin interferences in an enzymatic acetaminophen assay

Differences in bilirubin and biliverdin interferences in an enzymatic acetaminophen assay

Preparation of NiFe2O4/graphene nanocomposite and its application as a modifier for the fabrication of an electrochemical sensor for the simultaneous determination of tramadol and acetaminophen

An effective electrochemical sensor for the rapid and simultaneous determination of tramadol and acetaminophen based on carbon paste electrode (CPE) modified with NiFe2O4/graphene nanoparticles was developed. The structures of the synthesized NiFe2O4/graphene nanocomposite and the electrode composition were confirmed by X-ray diffraction (XRD) spectrometry, Fourier transform infrared (FT-IR) spectrometry and scanning electron microscopy (SEM). The peak currents of square wave voltammetry of tramadol and acetaminophen increased linearly with their concentration in the range of 0.01–9μmolL−1. The detection limit for their determination was found to be 0.0036 and 0.0030μmolL−1, respectively. The results show that the combination of graphene and NiFe2O4 nanoparticles causes a dramatic enhancement in the sensitivity of the sensor. The fabricated sensor exhibited high sensitivity and good stability, and would be valuable for the clinical assay of tramadol and acetaminophen.

Simultaneous Determination of Some Analgesic Drugs Using Mixed Mode Stationary Phase

A new HPLC method based on a mixed mode stationary phase Hypersil Duet C18/SAX was developed and applied for the simultaneous determination of acetaminophen, acetylsalicylic acid and codeine. Parameters, such as the composition of the mobile phase, the nature of the organic modifier, the buffer type and the flow rate were investigated to optimize the separation. The results obtained show that the new HPLC method is rapid, highly efficient and selective. The studied compounds are separated in 10 min, by means of a mobile phase containing phosphate buffer (pH 7.50) and methanol (65:35 v v−1). The retention mechanisms of each analyte were investigated using both the linear solvent strength theory and stoichiometric displacement model. The method was fully validated and showed good linearity for each compound for a concentration ranging between 2.0 and 40 μg mL−1. The limits of detection and quantification were determined and they are lower than 0.1 μg mL−1. The precision (RSD) of the method does not exceed 2 % for all studied compounds. The method was successfully applied for the assay of acetaminophen, acetylsalicylic acid and codeine in pharmaceutical formulations.

Validation Thin Layer Chromatography for the Determination of Acetaminophen in Tablets and Comparison with a Pharmacopeial Method

Adsorption thin layer chromatography (NP-TLC) with densitometry has been established for the identification and the quantification of acetaminophen in three leading commercial products of pharmaceutical tablets coded as brand: P1 (Product no. 1), P2 (Product no. 2), and P3 (Product no. 3). Applied chromatographic conditions have separated acetaminophen from its related substances, namely, 4-aminophenol and and 4′-chloroacetanilide. UV densitometry was performed in absorbance mode at 248 nm. The presented method was validated by specificity, range, linearity, accuracy, precision, detection limit, quantitative limit, and robustness. The TLC-densitometric method was also compared with a pharmacopeial UV-spectrophotometric method for the assay of acetaminophen, and the results confirmed statistically that the NP-TLC-densitometric method can be used as a substitute method. It could be said that the validated NP-TLC-densitometric method is suitable for the routine analysis of acetaminophen in quantity control laboratories.

Reliability of the reported ingested dose of acetaminophen for predicting the risk of toxicity in acetaminophen overdose patients

The present study examines the relationship between the dose of acetaminophen reported to have been ingested by patients and the occurrence of serum acetaminophen levels above the 'possible toxicity' line in patients presenting at the hospital after acetaminophen overdose. The prognostic value of patient-reported dosage cut-offs of 8, 10 and 12 g was determined. This retrospective cohort study included patients admitted to the emergency department or hospital within 24 hours of acetaminophen ingestion. Serum acetaminophen concentrations were considered to be the gold standard, and specificity, sensitivity and positive/negative predictive values were calculated from the reported ingested dose, to predict toxicity using the Rumack-Matthew nomogram (i.e. the 'possible toxicity' treatment line) and standard equations. Of 305 patients identified, 291 met the study inclusion criteria, and 121 (41.6%) had serum acetaminophen concentrations above the 'possible toxicity' treatment line. The range of patient-reported acetaminophen ingested was 1-75 g, with 185 patients (63.6%) reporting ≥8 g. One hundred eighteen patients (97.5%) who reported ingesting ≥8 g had serum acetaminophen concentrations above the '150-line', compared with only three patients (2.5%) who reported ingesting <8 g (p < 0.001). The positive predictive value of a patient-reported dose ≥8 g for predicting serum acetaminophen concentrations above the 'possible toxicity' treatment line was 63.78%, with a negative predictive value of 97.17%. The sensitivity of patient-reported doses ≥8 g was high (97.52%) but with low specificity (60.59%). The sensitivity of patient-reported doses ≥10 g also was high (89.26%) with low specificity (65.29%), whereas the sensitivity of ≥12 g dose was low (61.16%) with high specificity (86.47%). Patient-reported doses of acetaminophen are good risk indicators for acetaminophen overdose patients in Malaysia. Patient-reported ingestion of ≥8 g (as a cut-off dose) had a higher sensitivity than ≥10 g or ≥12 g. The results of this study have important implications for toxicity risk evaluations in areas with poor serum acetaminophen assay availability.