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Route 206 and Province Line Road, Lawrenceville, NJ 08543, USA RATIONALE: Research on disorders of the central nervous system (CNS) has shown that an imbalance in the levels of specific endogenous neurotransmitters may underlie certain CNS diseases. These alterations in neurotransmitter levels may provide insight into pathophysiology, but can also serve as disease and pharmacodynamic biomarkers. To measure these potential biomarkers in vivo, the relevant sample matrix is cerebrospinal fluid (CSF), which is in equilibrium with the brain's interstitial fluid and circulates through the ventricular system of the brain and spinal cord. Accurate analysis of these potential biomarkers can be challenging due to low CSF sample volume, low analyte levels, and potential interferences from other endogenous compounds. METHODS: A protocol has been established for effective method development of bioanalytical assays for endogenous compounds in CSF. Database searches and standard-addition experiments are employed to qualify sample preparation and specificity of the detection thus evaluating accuracy and precision. RESULTS: This protocol was applied to the study of the histaminergic neurotransmitter system and the analysis of histamine and its metabolite 1-methylhistamine in rat CSF. CONCLUSIONS: The protocol resulted in a specific and sensitive novel method utilizing pre-column derivatization ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS), which is also capable of separating an endogenous interfering compound, identified as taurine, from the analytes of interest.

A sensitive HPLC method using fluorescence detection was developed to determine kynurenic acid (KYNA) level in rat cerebrospinal fluid (CSF). The method development was accomplished by screening different columns, optimizing zinc acetate concentration and determining the optimal HPLC flow rate. This method allowed direct injection of the CSF samples onto an Xselect C18 column and KYNA levels were measured fluorometrically by forming a fluorescent complex with zinc acetate that was delivered post-column. The limit of quantitation was 0.2 n m with 30 μL injection, corresponding to 6 fmol (signal-to-noise ratio = 10). The improved sensitivity enabled the measurement of KYNA in naive and drug-treated rat CSF.

Acetyl-L-carnitine (ALCAR) is a potential biomarker for the modulation of brain neurotransmitter activity, but is also present in cerebrospinal fluid (CSF). Recent studies have utilized hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) based assays to detect and quantify ALCAR within biofluids such as urine, plasma and serum, using various sample pretreatment procedures. In order to address the need to quantify ALCAR in CSF on a high-throughput scale, a new and simple HILIC-MS/MS assay has been successfully developed and validated. For rapid analysis, CSF sample pretreatment was performed via 'dilute and shoot' directly onto an advanced HILIC column prior to MS/MS detection. This newly developed HILIC-MS/MS assay shows good recoveries of ALCAR without the need for chemical derivatization and multistep sample extraction procedures. The employment of this assay is suitable for the high-throughput bioanalysis and quantification of ALCAR within the CSF of various animal models and human clinical studies.

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