Cerebrospinal fluid/serum analysis

Analysis of small ions has been published for many applications other than pharmaceutical applications, and has a growing impact in industrial, environmental, biomedical, clinical, and forensic laboratories. Sample matrices range from simple tap water to Kraft black liquor, including river and seawater, beer and wine, environmental water, and nuclear plant water, but also body fluids such as serum, urine, plasma, cerebrospinal fluid, and many others. Those topics alone would require a separate book. 

Other antibiotics studied using RPC include chloramphenicol which could be determined at serum concentrations of about 0.5 /xg/ml using 0.1 ml samples ( 15) after extraction with ethyl acetate (316). A simple method for the analysis of chloramphenicol in serum and cerebrospinal fluid has been reported in which the analyte is extracted into methanol and the extract chromatographed with acetic acid-water methanol (1 62 37) as the mobile phase (i/7). 

Sterol analysis and quantification can be performed using plasma, serum, and cerebrospinal fluid (CSF) samples. Analysis of sterols from tissues and cells requires a pre-extraction work-up. The requirements for the different specimens are as follows ... 

In contrast, ProteinChip technology allows the detailed analysis of A(3 fragments and their modifications (22). Peptides are captured on arrays coated with anti-Ap Ab, washed to remove nonspecifically bound fragments, and finally detected by SELDI-TOF MS. The resolution of the technique allows the discrimination of peptides of similar mass and modified products, e.g., oxidized peptides versus native peptides, which differ by only 16 Da. ProteinChip technology has been used extensively to detect Af> fragments in various samples, including cell culture supernatants, serum, plasma, and cerebrospinal fluid (CSF) (21-23). 

The measurement of Zn in body tissues or fluids for clinical purposes does not require the sensitivity of ETA—AAS. The very early methods using carbon-rod atomisers required only 0.5 pi serum for direct analysis of Zn [48], and current techniques require <0.1 pi. The measurement of Zn in cerebrospinal fluid (CSF) by ETA—AAS [49] could have just as easily been determined using pulsed —nebulisation FAAS with <100 pi sample volumes. 

The bioanalyst can be required to analyse most biofluids although the most common are urine and the aqueous phase of blood, i.e. plasma or serum. Other samples may be cell and tissue extracts, synovial fluid, cerebrospinal fluid (CSF) and saliva. In the case of urine and CSF with their very low protein content it might be possible to directly inject the sample into an HPLC column. With most silica-based packing materials, direct injection of blood proteins will rapidly lead to column deterioration. HPLC columns are expensive and their efficiency is easily lost so correct preparation of samples will not only improve column life but also improve the results. At its simplest it is only necessary to remove particulate matter from samples to prevent clogging of the column and frits. Modern HPLC packings are very susceptible to contamination by proteins, fats and other macromolecules from biological samples and it is necessary to remove these (except of course for protein analysis). 

In most applications, the bioanalysis involves the analysis of a number of dmgs, or one dmg and (some of) its metabolites in biological fluids, especially whole blood, plasma, serum, or urine. However, other matrices are studied as well various tissues (skin, liver, brain, thyroid gland), faeces, hair, tear fluid, cerebrospinal fluid, semen. In most studies, the analysis of samples from human origin or from rats is performed, although the analysis of samples from rabbits, mice, minipigs, dogs, and monkeys is also performed. 

The determination of ddl [44] and d4T [45] in human serum was reported by online SPE-LC-MS with positive-ion ESI-MS. 3TC and ddl were applied as IS. The analysis time was less than 5 min for both methods. The LOQ was 10 ng/ml for both compounds. Another group described the determination of ddl and d4T in human plasma, bronchoalveolar lavage fluid (BALE), alveolar cells, PBMC, seminal plasma, cerebrospinal fluid (CSF), and tonsil tissue [57]. Depending on the matrix, either isocratic or gradient LC was applied after SPE sample pretreatment. Positive-ion ESI-MS in SRM mode was applied. The LOQ for both compounds were 2.0 ng/ml in plasma, 0.5 ng/ml in CSF, 0.4 ng/ml in alveolar cells, BALF, and PBMC, 1 ng/ml in seminal plasma, and 0.01 ng/mg in tonsil tissue. 

As already described, the original sample is of importance. Serum or plasma can be employed with all systems. In the case of plasma, attention must be paid to the anticoagulant, since it may interfere with the analysis. So far, reference ranges for analytes measured in whole blood are known for a few parameters only. Other body fluids, such as urine or cerebrospinal fluid, can be used only with the Ektachem system and a few reagent carriers of the Reflotron system. The majority of reagent carriers or slides are only suitable for serum, plasma or whole blood. The use of dry chemistry in veterinary medicine poses special problems although results have been published, information is still lacking. 

Banters et al. (2003) demonstrated Cryptococcus neoformans in cerebrospinal fluid (CSF) and serum. Their 30-min procedure was based on the non-specific labelling with ChemChrome V3 in combination with a second analysis using immunofluorescence. To that end, cells were labelled with a specific primary antibody against a capsular polysaccharide and a secondary antibody conjugated with FITC. 

Ritchie, S., Comprehensive metabolomic profiling of serum and cerebrospinal fluid Understanding disease, human variability, and toxicity, in Surrogate Tissue Analysis, 1st edition, Burczynski, M.E., and Rockett, J.C., Eds., CRC Press LLC, Boca Raton, FL, 2006, p. 165. 

The specimen volume needed for analysis is minimal (e.g., 5pL), the dead volume in a specimen container is minimized, and the instrument has a smart probe as described above. Evaporation control is possible with lids of some kind, and a variety of different types of specimens, e.g., serum, plasma, urine, cerebrospinal fluid, and other body fluids can be accommodated on the same instrument. Obviously, the menu of enzyme tests must be broad enough to consolidate the testing on as few work stations as possible. 

The analyte may be present in a variety of matrices such as plasma, serum, cerebrospinal fluid (CSF), urine, or other biological fluid, all of which contain a multitude of interfering factors that can impact the method s performance. As mentioned earlier in the chapter, LBA samples are not pretreated prior to analysis, thus calibrators and quality control (QC) samples should also be prepared in the study matrix to best mimic these samples and ensure accurate measurements. The ideal matrix (1) has low background signal in the assay (OD <0.1 for chromogenic end points), (2) has minimal or no analyte-like activity, (3) is devoid of interfering factors, and (4) demonstrates a response that is proportional to the concentration of the spiked analyte. Access to a prototype method using an assay buffer of defined composition as a reference helps to identify an appropriate matrix. 

The main clinical significance of cerebrospinal fluid (CSF) protein eleetrophoresis is for the detection of the oligoclonal bands, which are present in multiple sclerosis in the gamma region. Similar to urine, proteins in the CSF are present fluid in very low concentration (100 times less than serum). For the majority of the samples, a 10- to 20-fold concentration is preferred before analysis by CE (by the same membrane concentrators used for urine). CSF protein separation can be accomplished in less than 10 min with CE versus 2 h for AG with the ability to detect oligoclonal banding by this technique [36]. 

Unlike mass spectroscopy, gel electrophoresis does not provide a quantitative value for the amount of given protein. However, it provides a low cost and relatively rapid method for the analysis of multiple proteins in a specimen, especially when implemented as a capillary electrophoresis system. Therefore, it has been used for the separation of enzymes (e.g., creatinine phosphokinase), mucopolysaccharides, plasma, serum, cerebrospinal fluid, urine, and other bodily fluids [13]. It is also used for quality control applications for the manufacturing of biological compounds to verify the purity or to examine the manufacturing yield [14]. 

---