Quantitative tandem mass spectrometry in the clinical laboratory: Regulation and opportunity for validation of laboratory developed tests.

Tandem mass spectrometry is an important analytical tool for clinical laboratories, but tests developed and validated in-house (laboratory developed tests, or LDTs) require special consideration. In late 2022, the forecast for United States (U.S.) federal regulation of LDTs changed unexpectedly when the VALID Act was not passed by the U.S. Congress. This Act would have modified the Food and Drug Administration's (FDA's) role to increase regulatory oversight for LDT providers. In this revised context, we review optimization of quantitative mass spectrometry LDT validation and suggest avenues other than an additional FDA mandate to achieve uniform best practice. Common challenges, logistical barriers, and recommendations for easing the burden of best-quality quantitative mass spectrometry LDT method validation are discussed.

Quantitation of Aldosterone in Serum or Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate determination of serum and plasma aldosterone is essential for screening, diagnosis, and subtype classification of primary aldosteronism (PA). Its measurement is also used in the investigation of adrenal incidentaloma, adrenal carcinoma, Addison's disease, congenital adrenal hyperplasia, renal artery stenosis, and renal tubular channelopathies. We describe a simple and robust method for the accurate and precise measurement of aldosterone in serum or plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). After addition of internal standard, aldosterone is extracted from serum samples using supported liquid extraction (SLE) with methyl tert-butyl ether (MtBE). The MtBE is evaporated to dryness, and the sample is reconstituted with mobile phase before injection onto the LC-MS/MS and quantitation using an eight-point calibration curve. The assay calibration range is approximately 50-6500 pM (0.16-234 ng/dL) with total imprecision between 6.8% and 4.1% for concentrations between about 50 and 1000 pM, respectively.

Quantitation of IgG Subclasses in Serum Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Serum IgG subclasses (IgGSC) are measured for a number of indications, but the most common are the identification of selective immunodeficiency disease and the diagnosis of IgG4-related disease (IgG4RD). Traditional nephelometric (IN) assays can suffer from two issues impacting the accuracy of the results: (1) hook effect and (2) antibody cross-reactivity between the subclasses. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not vulnerable to these modes of interference and therefore serves as an excellent and relatively inexpensive means of diagnosing and/or monitoring the relevant clinical conditions.We describe a semiautomated and simple method for the accurate and precise measurement of IgGSC from 20 µL of serum using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method following digestion of serum proteins in 96-well plate format. Due to the high abundance of the target proteins, no specialized sample preparation (such as solid phase extraction) is required. Twenty microliters are injected to the LC-MS/MS system. Quantitation is performed against a five-point duplicate linear calibration curve prepared in blank matrix. The assay calibration range is 0.38-7.74 g/L for IgG1, 0.24-4.46 g/L for IgG2, 0.038-0.752 g/L for IgG3, 0.025-0.435 g/L for IgG4, and 0.62-15.5 g/L for total IgG. Total IgG is used as an internal quality control marker and is compared to the sum of the four subclass results. Total imprecision in clinical production has been observed to be 5.1-10.6% for in-house prepared control materials having IgGSC mean values in the range of 0.38-8.43 g/L for IgG1, 0.22-3.76 g/L for IgG2, 0.0387-0.721 g/L for Ig3, and 0.0279-1.46 g/L for IgG4. Limit of quantitation (LoQ) was determined to be 0.29 g/L for IgG1, 0.22 g/L for IgG2, 0.019 g/L for IgG3, and 0.0067 g/L for IgG4.

Quantitation of Renin Activity in Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate determination of plasma renin activity (PRA) is essential for the development and maintenance of an effective screening program for primary aldosteronism (PA). PRA measurement can also be useful in the investigation of renal artery stenosis, syndrome of mineralocorticoid excess, Addison's disease, congenital adrenal hyperplasia, Bartters and Gitelman syndromes, and for inherited defects in the renin angiotensin aldosterone system (RAAS). We describe a semiautomated and simple method for the accurate and precise measurement of PRA from 500 µL of plasma (250 µL if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3 h AngI generation step at 37 °C in buffering conditions at pH 6, the reaction is quenched with 10% formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/MS system. Quantitation is performed against a 7-point calibration linear curve prepared in buffer. The assay calibration range is 0.34-30.0 ng/mL, which corresponds to PRA values of 0.11-10.0 ng/mL/h: much wider than was possible using traditional competitive antibody-based methods. Total precision in clinical production has been observed to be 5.8-5.0% for BioRad Hypertension Control materials having nominal PRA values ranging from 1.73 to 12.43 ng/mL/h. At AngI concentrations of 0.06 ng/L (corresponding to a PRA of 0.02 ng/mL/h), signal-to-noise ratio is 50:1, indicating that the limit of quantitation is well below the level required for clinical use.

Quantitation of Thyroglobulin in Serum Using SISCAPA and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate measurement of thyrogloblulin (Tg) at low concentrations is essential for recurrence-monitoring in patients who have been treated for papillary and follicular thyroid cancers. The immunoassays commonly employed by clinical laboratories to measure Tg are known to suffer interferences from thyroglobulin autoantibodies (TgAb).We describe a semiautomated stable isotope standards and capture by antipeptide antibodies (SISCAPA®) LC-MS/MS method for the accurate and precise measurement of Tg using 400 uL of serum. Following trypsin digestion of serum proteins in a 96-well plate format, a Tg-specific peptide is captured and concentrated using a monoclonal antibody bound to protein G-coated paramagnetic beads. Eighteen microliters of concentrate are injected into the LC-MS/MS system. Quantitation is performed against a 6-point linear calibration curve prepared in a blank matrix. The assay calibration range is 0.1-10 ng/mL, the range of clinical interest for recurrence detection. Total imprecision in clinical production has been observed to be 13.8% and 6.54% for in-house prepared control materials having Tg concentrations of 0.24 ng/mL and 0.94 ng/mL, respectively. Limit of quantitation was determined to be 0.1 ng/mL.

Establishing pre-analytical requirements and maximizing peptide recovery in the analytical phase for mass spectrometric quantification of amyloid-ß peptides 1-42 and 1-40 in CSF.

OBJECTIVES: Amyloid-ß (Aß) peptides in cerebrospinal fluid (CSF), including Aß42 (residues 1-42) and Aß40 (residues 1-40), are utilized as biomarkers in the diagnostic workup of Alzheimer's disease. Careful consideration has been given to the pre-analytical and analytical factors associated with measurement of these peptides via immunoassays; however, far less information is available for mass spectrometric methods. As such, we performed a comprehensive evaluation of pre-analytical and analytical factors specific to Aß quantification using mass spectrometry. METHODS: Using our quantitative mass spectrometry assay for Aß42 and Aß40 in CSF, we investigated the potential for interference from hemolysate, bilirubin, lipids, and anti-Aß-antibodies. We also optimized the composition of the calibrator surrogate matrix and Aß recovery during and after solid phase extraction (SPE). RESULTS: There was no interreference observed with total protein up to 12 g/L, hemolysate up to 10% (v/v), bilirubin up to 0.5% (v/v), intralipid up to 1% (v/v), or anti-Aß-antibodies at expected therapeutic concentrations. For hemolysate, bilirubin and lipids, visual CSF contamination thresholds were established. In the analytical phase, Aß recovery was increased by ∼50% via SPE solvent modifications and by over 150% via modification of the SPE collection plate, which also extended analyte stability in the autosampler. CONCLUSIONS: Attention to mass spectrometric-specific pre-analytical and analytical considerations improved analytical sensitivity and reproducibility, as well as, established CSF specimen acceptance and rejection criteria for use by the clinical laboratory.

Continuous reference intervals for pediatric testosterone, sex hormone binding globulin and free testosterone using quantile regression.

Testosterone (T), sex hormone binding globulin (SHBG), free testosterone (FT) and bioavailable testosterone (BAT) are commonly employed tests in pediatric endocrinology and all require age-dependent reference intervals for interpretation. The common methods used to derive these reference intervals require decisions about data shape and/or age partition thresholds, which can result in sharp differences between age groups, particularly for pubescent children. Partitioning also results in a form of data loss, where data from one age-bin is completed disconnected from the adjacent age-bins. Non-parametric continuous reference intervals methods have previously been developed to avoid some of these drawbacks. These strategies use all the available data and smooth transitions between ages avoiding partitioning. However, the fitting process involves selection and adjustment of many parameters and it can be difficult to maintain a reproducible approach. Here we provide a workflow for non-parametric continuous reference intervals applied to T, FT, BAT, and SHBG using the R language quantregGrowth package. T measurements were determined by LC-MS/MS, FT and BAT were calculated, and SHBG was measured on the Roche Cobas e601. The continuous interval methodology is described in detail with code examples and illustrations for reproducibility.

An automated clinical mass spectrometric method for identification and quantification of variant and wild-type amyloid-ß 1-40 and 1-42 peptides in CSF.

INTRODUCTION: We developed an automated liquid chromatography-tandem mass spectrometry high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for multiplex quantification of wild-type (wt) amyloid ß (Aß) peptides 1-40 (Aß40) and 1-42 (Aß42) and detection of variant Aß peptides in cerebrospinal fluid. METHODS: The multiplex Aß HPLC-MS/MS assay was validated in a clinically accredited laboratory following regulatory guidelines, with Aß42 calibration assigned to the ERM/IFCC certified reference material; sequence variants were additionally multiplexed into the method. RESULTS: Sample preparation was fully automated on a liquid handler. The assay quantified wt-Aß42 and wt-Aß40 and detected sequence variants, when present, within the Aß42 sequence. DISCUSSION: Extension of the HPLC-MS/MS approach for quantification of wt-Aß42 and wt-Aß40 to include known sequence variants increases analytical accuracy of the mass spectrometric approach and enables identification of cases of autosomal dominant Alzheimer's disease. Development of an automated workflow and selection of appropriate instrumentation enabled deployment of this method in routine clinical testing.

Comparison of four clinically validated testosterone LC-MS/MS assays: Harmonization is an attainable goal.

INTRODUCTION: Immunoassays and liquid chromatography-tandem mass spectrometry assays are commonly employed in clinical laboratories for measurement of total testosterone in serum. Results obtained from either of these methodologies compare poorly due to differences in calibration and/or inadvertent detection of interfering substances by the immunoassays. Standardization efforts are underway, but recent studies indicate that accuracy remains an issue. METHODS: This study compares the results from four independently developed and validated LC-MS/MS assays for total testosterone. The calibration for each assay was verified using National Institute of Standards and Technology Standard Reference Material 971. RESULTS: Initially, one of the four assays had a mean percent difference of +11.44%, compared to the All Method Mean, but following re-verification of all five non-zero calibrator concentrations with the NIST SRM 971, the mean percent difference decreased to -4.88%. Subsequently, the agreement between all four assays showed a mean bias of <5% across the range of all testosterone concentrations (0.13-38.10 nmol/L; 3.7-1098 ng/dL), including at low concentrations of <1 nmol/L (<29 ng/dL). CONCLUSIONS: Excellent agreement between four independently developed LC-MS/MS assays demonstrates that harmonization using standard reference material is attainable. However, as we found in this study, to ensure accurate calibration it is critical to validate the concentrations of new lots of calibrators.

An automated mass spectrometric blood test for therapeutic drug monitoring of infliximab.

Infliximab is a monoclonal antibody therapy used to treat several chronic immune-mediated diseases, including Crohn's disease, ulcerative colitis, and rheumatoid arthritis. Infliximab acts by binding to tumor necrosis factor and, thus, inhibiting the inflammatory cascade. While it is a highly effective therapy, a subset of patients on infliximab will develop a loss of response to therapy. In these circumstances, therapeutic drug monitoring of infliximab offers a rational approach to clinical decision making and is associated with improved outcomes. While infliximab has most commonly been measured by immunometric approaches, mass spectrometric approaches offer the opportunity to improve test accuracy and reduce test costs. Herein, we describe a simple, bottom-up high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) approach for quantitation of infliximab in serum. Method development included pre-digestion and digestion experiments to determine critical sample preparation steps, optimization of the workflow and selection of rapidly produced proteolytic peptide(s) for quantitation. The workflow was further improved by automating all sample preparation steps on a robotic liquid handler, facilitating implementation in routine clinical use. A method comparison was performed against a Health Canada and US Food and Drug Administration licensed enzyme-linked immunosorbent assay. Our LC-MS/MS assay accurately reported concentrations based on drug manufacturer targets and demonstrated no interference from endogenous antibodies to infliximab; immunoassay methods did not share these performance characteristics. This LC-MS/MS method provides a workflow amenable to implementation in a clinical laboratory and desired performance characteristics for guiding clinical decision making.

Rivaroxaban and apixaban induce clotting factor Xa fibrinolytic activity.

Essentials Activated clotting factor X (FXa) acquires fibrinolytic cofactor function after cleavage by plasmin. FXa-mediated plasma fibrinolysis is enabled by active site modification blocking a second cleavage. FXa-directed oral anticoagulants (DOACs) alter FXa cleavage by plasmin. DOACs enhance FX-dependent fibrinolysis and plasmin generation by tissue plasminogen activator. BACKGROUND: When bound to an anionic phospholipid-containing membrane, activated clotting factor X (FXa) is sequentially cleaved by plasmin from the intact form, FXaα, to FXaß and then to Xa33/13. Tissue-type plasminogen activator (t-PA) produces plasmin and is the initiator of fibrinolysis. Both FXaß and Xa33/13 enhance t-PA-mediated plasminogen activation. Although stable in experiments using purified proteins, Xa33/13 rapidly loses t-PA cofactor function in plasma. Bypassing this inhibition, covalent modification of the FXaα active site prevents Xa33/13 formation by plasmin, and the persistent FXaß enhances plasma fibrinolysis. As the direct oral anticoagulants (DOACs) rivaroxaban and apixaban bind to the FXa active site, we hypothesized that they similarly modulate FXa fibrinolytic function. METHODS: DOAC effects on fibrinolysis and the t-PA cofactor function of FXa were studied in patient plasma, normal pooled plasma and purified protein experiments by the use of light scattering, chromogenic assays, and immunoblots. RESULTS: The plasma of patients taking rivaroxaban showed enhanced fibrinolysis correlating with FXaß. In normal pooled plasma, the addition of rivaroxaban or apixaban also shortened fibrinolysis times. This was related to the cleavage product, FXaß, which increased plasmin production by t-PA. It was confirmed that these results were not caused by DOACs affecting activated FXIII-mediated fibrin crosslinking, clot ultrastructure and thrombin-activatable fibrinolysis inhibitor activation in plasma. CONCLUSION: The current study suggests a previously unknown effect of DOACs on FXa in addition to their well-documented anticoagulant role. By enabling the t-PA cofactor function of FXaß in plasma, DOACs also enhance fibrinolysis. This effect may broaden their therapeutic indications.

Quantitation of Aldosterone in Serum or Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate determination of serum and plasma aldosterone is essential for screening, diagnosis, and subtype classification of primary aldosteronism (PA). Its measurement is also used in the investigation of adrenal incidentaloma, adrenal carcinoma, Addison's disease, congenital adrenal hyperplasia, renal artery stenosis, and renal tubular channelopathies. We describe a simple and robust method for the accurate and precise measurement of aldosterone in serum or plasma using liquid chromatography and tandem mass spectrometry (LC-MS/MS). After addition of internal standard, aldosterone is extracted from serum samples using supported liquid extraction (SLE) with methyl-t-butyl ether (MtBE). The MtBE is evaporated to dryness and sample is reconstituted with mobile phase before injection onto the LC-MS/MS and quantitation using an 8-point calibration curve. The assay calibration range is approximately 50-6500 pmol/L (0.16-234 ng/dL) with total imprecision between 6.8 and 4.1 % for concentrations between about 50 and 1000 pmol/L respectively.

Quantitation of Insulin Analogues in Serum Using Immunoaffinity Extraction, Liquid Chromatography, and Tandem Mass Spectrometry.

Insulin analysis is used in combination with glucose, C-peptide, beta-hydroxybutyrate, and proinsulin determination for the investigation of adult hypoglycemia. The most common cause is the administration of too much insulin or insulin secretagogue to a diabetic patient or inadequate caloric intake after administration of either. Occasionally there is a question as to whether hypoglycemia has been caused by an exogenous insulin-whether by accident, intent, or even malicious intent. While traditionally this was confirmed by a low or undetectable C-peptide in a hypoglycemic specimen, this finding is not entirely specific and would also be expected in the context of impaired counter-regulatory response, fatty acid oxidation defects, and liver failure-though beta-hydroxybutyrate levels can lend diagnostic clarity. For this reason, insulin is often requested. However, popular automated chemiluminescent immunoassays for insulin have distinctly heterogeneous performance in detecting analogue synthetic insulins with cross-reactivities ranging from near 0 % to greater than 100 %. The ability to detect synthetic insulins is vendor-specific and varies between insulin products. Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS) offers a means to circumvent these analytical issues and both quantify synthetic insulins and identify the specific type. We present an immunoaffinity extraction and LC-MS/MS method capable of independent identification and quantitation of native sequence insulins (endogenous, Insulin Regular, Insulin NPH), and analogues Glargine, Lispro, Detemir, and Aspart with an analytical sensitivity for endogenous insulin of between 1 and 2 µU/mL in patient serum samples.

Quantitation of Plasma Renin Activity in Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate determination of plasma renin activity (PRA) is essential for the development and maintenance of an effective screening program for primary aldosteronism (PA). PRA measurement can also be useful in the investigation of renal artery stenosis, syndrome of mineralocorticoid excess, Addison's disease, congenital adrenal hyperplasia, Bartter and Gitelman syndromes, and for inherited defects in the renin angiotensin aldosterone system (RAAS). We describe a semi-automated and simple method for the accurate and precise measurement of PRA from 500 µL of plasma (250 µL if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3 h AngI generation step at 37 °C in buffering conditions at pH 6, the reaction is quenched with 10 % formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/MS system. Quantitation is performed against a 7-point calibration linear curve prepared in buffer. The assay calibration range is 0.34-30.0 ng/mL which corresponds to PRA values of 0.11-10.0 ng/mL/h: much wider than was possible using traditional competitive antibody-based methods. Total precision in clinical production has been observed to be 5.8 to 5.0 % for Bio-Rad Hypertension Control materials having nominal PRA values ranging from 1.73 to 12.43 ng/mL/h. At AngI concentrations of 0.06 ng/L (corresponding to a PRA of 0.02 ng/mL/h), signal to noise ratios are 50:1 indicating that the limit of quantitation is well below the level required for clinical use.

An automated assay for the clinical measurement of plasma renin activity by immuno-MALDI (iMALDI).

Plasma renin activity (PRA) is essential for the screening and diagnosis of primary aldosteronism (PA), a form of secondary hypertension, which affects approximately 100 million people worldwide. It is commonly determined by radioimmunoassay (RIA) and, more recently, by relatively low-throughput LC-MS/MS methods. In order to circumvent the negative aspects of RIAs (radioisotopes, cross-reactivity) and the low throughput of LC-MS based methods, we have developed a high-throughput immuno-MALDI (iMALDI)-based assay for PRA determination using an Agilent Bravo for automated liquid handling and a Bruker Microflex LRF instrument for MALDI analysis, with the goal of implementing the assay in clinical laboratories. The current assay allows PRA determination of 29 patient samples (192 immuno-captures), within ~6 to 7h, using a 3-hour Ang I generation period, at a 7.5-fold faster analysis time than LC-MS/MS. The assay is performed on 350µL of plasma, and has a linear range from 0.08 to 5.3ng/L/s in the reflector mode, and 0.04 to 5.3ng/L/s in the linear mode. The analytical precision is 2.0 to 9.7% CV in the reflector mode, and 1.5 to 14.3% CV in the linear mode. A method comparison to a clinically employed LC-MS/MS assay for PRA determination showed excellent correlation within the linear range, with an R(2) value of ≥0.98. This automated high throughput iMALDI platform has clinically suitable sensitivity, precision, linear range, and correlation with the standard method for PRA determination. Furthermore, the developed workflow based on the iMALDI technology can be used for the determination of other proteomic biomarkers. This article is part of a Special Issue entitled: Medical Proteomics.

Mouse strain differences in autonomic responses to stress.

In humans, anxiety disorders are often accompanied by an overactive autonomic nervous system, reflected in increased body temperature (BT) and heart rate (HR). In rodents, comparable effects are found after exposure to stress. These autonomic parameters can give important information on stress and anxiety responses in mice. In the present experiments, stress reactivity of three frequently used mouse strains [129 Sv/Ev, Swiss Webster (SW) and C57 BL/6] was assessed using their autonomic stress responses. BT, HR and activity were telemetrically measured. Undisturbed circadian rhythms already showed clear differences between the mouse strains. Hereafter, autonomic responses to stressors with increasing intensity were measured. Strain differences were found in magnitude and duration of the stress responses, especially after high-intensity stressors. Generally, C57BL/6 mice showed the largest autonomic response, SW the lowest and the 129Sv/Ev the intermediate response. Interestingly, the observed ranking in autonomic stress response does not match the behavioral stress responsivity of these strains. Finally, sensitivity to the anxiolytic diazepam (0, 1, 2, 4 and 8 mg/kg) was tested using the stress-induced hyperthermia paradigm. Pharmacological sensitivity to diazepam differed between the strains with the 129Sv/Ev being most sensitive. These studies show that simultaneous measurement of behavioral and autonomic parameters under stressful conditions contributes considerably to a better interpretation of anxiety and stress levels in mice.

Urocortin expression in the Edinger-Westphal nucleus is down-regulated in transgenic mice over-expressing neuronal corticotropin-releasing factor.

In recent years a large body of evidence has emerged linking chronic stress with increased vulnerability for depression and anxiety disorders. As corticotropin-releasing factor (CRF) is hypersecreted under these psychological conditions, we used our CRF-overexpressing (CRF-OE) mouse line to study underlying brain mechanisms possibly causing these disorders. Urocortin (Ucn), a recently discovered member of the CRF peptide family may play a role in the pathophysiology of stress-induced disorders. Stressors recruit Ucn-immunoreactive neurons in the Edinger-Westphal nucleus (E-WN), which is the major site of Ucn expression. Furthermore, E-WN Ucn mRNA levels are upregulated in CRF-deficient mice. Based on these findings, we hypothesized the down-regulation of E-WN Ucn in CRF-OE mice and consequently, altered responsiveness to stressful stimuli. Our results support this hypothesis as we found weaker immunohistochemical labeling with anti-Ucn and a six times weaker Ucn mRNA signal in E-WN in CRF-OE mice. Moreover, E-WN Ucn-expressing neurons mounted a response to acute challenge in CRF-OE mice too. From these results it is concluded that the CRF and E-WN Ucn neuronal systems work in concert in response to acute challenges, but are inversely regulated in their activities during chronic hyperactivity of the hypothalamo-pituitary-adrenal axis.

Effects of methylphenidate, desipramine, and L-dopa on attention and inhibition in children with Attention Deficit Hyperactivity Disorder.

The objective of this study was to investigate the effects of methylphenidate (MPH) on attention and inhibition in children with Attention Deficit Hyperactivity Disorder (ADHD) and to establish what the relative contributions of the noradrenergic and dopaminergic systems to this effect were. In addition to MPH, two other drugs were administered in order to affect both transmitter systems more selectively, L-dopa (dopamine (DA) agonist) and desipramine (DMI) (noradrenaline (NA) re-uptake inhibitor). Sixteen children with ADHD performed a stop-task, a laboratory task that measures the ability to inhibit an ongoing action, in a double-blind randomized within-subjects design. Each child received an acute clinical dose of MPH, DMI, L-dopa, and placebo; measures of performance and plasma were determined. The results indicated that inhibition performance was improved under DMI but not under MPH or L-dopa. The response-time to the stop-signal was marginally shortened after intake of DMI. MPH decreased omission and choice-errors and caused faster reaction times to the trials without the stop-tone. No effects of L-dopa whatsoever were noted. Prolactin levels were increased and 5-HIAA levels were lowered under DMI relative to placebo. It is suggested that the effects of MPH on attention are due to a combination of noradrenergic and dopaminergic mechanisms. The improved inhibition under DMI could be serotonergically mediated.