Quantitative clinical metabolite analyses

The use of SRM methods for quantitative bioanalysis represents increased dimensions of mass spectrometry analysis. SRM methods that use APCI-LC/MS/MS for the quantitative analysis of an antipsychotic agent, clozapine, in human plasma were described by Dear and co-workers (Dear et al., 1998). Preclinical development studies of clozapine in rats and dogs used HPLC with fluorescence detection (FLD). With this method, a better limit of quantitation (LOQ) of 1 ng/mL was obtained. As the compound moved into the clinical stages of development, a more sensitive method of analysis was required to obtain rapid metabolic information in support of drug safety evaluation studies. A standard LC/MS/MS method is used for the quantitative analysis of clozapine (I) and four metabolites (II-V) in human plasma (Figure 6.34). 

One of the first applications of HPLC in the clinical field was the quantitation of theophylline in asthmatic infants. This highly accurate measurement was an important test because of the very low amount of sample required and the accuracy of the determination (see Fig. 1-10). More recent clinically related HPLC separations include drugs and drug metabolites, neurochemicals and their metabolites, histamines, thyroid hormones, and enkephalins. The earliest bioresearch applications of HPLC included the determination of peptides, proteins, and amino acids. Application of HPLC to the analysis of these compounds remains important, as indicated by the rapid growth in references (Fig. 1-11). Bioresearch remains one of the most rapidly expanding growth areas of LC. 

According to current clinical practice, only a small part of the information obtained from examination of tissues and biological fluids is used. For disorders of metabolic pathways, reflected in the qualitative and quantitative composition of metabolites of endogenous compounds, analysis of the results of these examinations can be the basis for verification of hypotheses on the causes of such diseases. 

In the first instance, it is used to confirm a diagnosis when dealing with a patient with a metabolic problem. When the routine chemical screens indicate the presence of abnormal metabolites or raised levels of the usual metabolites, then MS can positively confirm their identity and thus a diagnosis, or, should it be a new inborn error, point to the metabolic pathways which are affected. In many of the organic acidurias described, the defect leads to gross excretion of one or more compounds and, as quantitative extraction is not necessary for their detection, simple solvent or other methods are normally used. In some instances it is vital that analysis be carried out quickly so as to indicate the most suitable clinical management of an acutely ill patient. 

LC-MS determination of drugs and metabolites in clinical studies is a key function in the success and effectiveness of clinical development of drugs. LC-MS has established an obvious advantage for quantitative pharmacokinetic studies in terms of analysis time, cost, and sample throughput. LC-MS/MS has powerful capacity in quantity bioanalysis. LC-MS in clinical applications such as in the diagnosis of inherited and acquired metabolic disorders, gastrointestinal disorders, cancer and diabetes, and therapeutic dmg monitoring is still frequently required. 

In addition to their clinical value in the symptomol-ogy of tissue damage, E. are exploited as reagents for the specific qualitative and quantitative analysis of other metabolites, such as glucose, ATP, lactate, etc. Certain proteases are used medically to aid poor digestion, to prevent blood coagulation and to promote fibrinolysis... 

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