Liquid chromatography mass spectrometry measurements

The use of direct UV spectrophotometry to measure sample concentrations in pharmaceutical research is uncommon, presumably due to the prevalence and attractiveness of high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) methods. Consequently, most researchers are unfamiliar with the value of UV detection, mainly that it is generally much faster than other methods - a very important asset in high-throughput screening. 

Bowers LD, SanauUah. 1996. Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometry. J Chromatogr B 687 61-68. 

Hayward, M. J., Hargiss, L. O., Munson, J. L., Mandiyan, S. P. and Wennogle, L. P., Validation of Solubility Measurements Using Ultra-Filtration Liquid Chromatography Mass Spectrometry (UP-LC/MS), American Society for Mass Spectrometry 2000 Conference Abstract, Long Beach, CA, USA, 2000. 

Jenkins et al. developed a capillary electrophoresis system for the measurement of iohexol as a marker of the glomerular filtration rate (GFR) with a run time of 5.25 min and a coefficient of variation (CV) of 4.3% at 80 mg L" [121]. The GFR, calculated from the plasma clearance, had a reproducibility of 5.47 %. A similar approach (liquid chromatography-mass spectrometry with positive electrospray ionization after enrichment by solid phase extraction) was applied by Putschew et al. for the determination of iodinated contrast agents in treatment plant effluents and surface waters [118]. 

J. C. Wolff, L. Barr and P. Moss, New ultraviolet signal actuated switching valve for the measurement of low level impurities by liquid chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry, 1999,13(23), 2376-2381. 

D Abramson. Measurement of ochratoxin A in barley extracts by liquid chromatography-mass spectrometry. J Chrom 391 315-320, 1987. 

Jaruga P, Rodriguez H, Dizdaroglu M (2001) Measurement of 8-hydroxy-2 -deoxyadenosine in DNA by liquid chromatography/mass spectrometry. Free Rad Biol Med 31 336-344 Johansen l,Boye E (1975) Radiation-induced DNA strand breaks in E. coli measured within a fraction... 

To establish a sensitive and specific liquid chromatography-mass spectrometry (time-of-flight) [LC-MS (TOF)] method for the determination of donepezil in human plasma after an oral administration of 5 mg donepezil hydrochloride tablet [29]. Alkalized plasma was extracted with isopropa-nol-n-hexane (3 97) and loratadine was used as internal standard (IS). Solutes were separated on a Cis column with a mobile phase of metha-nokacetate buffer (pH 4.0) (80 20). Detection was performed on a TOF mass spectrometry equipped with an electrospray ionization interface and operated in positive-ionization mode. Donepezil quantitation was realized by computing the peak area ratio (donepezil-loratadine) (donepezil m/z 380 [M + H]+ and loratadine m/z 383[M + H]+) and comparing them with calibration curve (r = 0.9998). The linear calibration curve was obtained in the concentration range of 0.1-15 jUg/1. The detection limit of donepezil was 0.1 /zg/1. The average recovery was more than 90%. The intra- and inter-run precision was measured to be below 15% of RSD... 

An example is the amount-of-substance fraction of glycated haemoglobin Ale in total haemoglobin measured by high-pressure liquid chromatography-mass spectrometry [8] and a related BCR certified reference material 405 [9],... 

Xu X, Veenstra TD, Fox SD, Roman JM, Issaq HJ, Falk R, Saavedra JE, Keefer LK, Ziegler RG (2005) Measuring fifteen endogenous estrogens simultaneously in human urine by high-performance liquid chromatography-mass spectrometry. Anal Chem 77 6646-6654... 

Nelson RE, Grebe SK, O Kane DJ, Singh RJ (2004) Liquid chromatography mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma. Clin Chem 50 373-384... 

Other purified enzyme assays include quantitative measurements of oxygen consumption with a Clark electrode in prostaglandin biosynthesis [108], radioimmunoassay (RIA) [109], enzyme immunoassay (EIA). [110,111], and a pulsed ultrafiltration liquid chromatography-mass spectrometry (LC-MS) binding assay [112],... 

Often there is a need for structural identification of unknowns without available reference compounds and the identification can be done in connection with the chromatographic separation. One approach is to run measurements directly on-line using HPLC as the separation technique with UV-detection and monitoring at several wavelengths, but this is often not enough for safety identification. The last 15 years have seen a rapid development of combined liquid chromatography-mass spectrometry instrumentation, and this technique is the most valuable tool in qualitative analysis today (se below). In the absence of a reference compound some unknown substances e.g., isomers of the desired compound may require NMR for their definitive identification. 

Atomic absorption spectrometry with flame (AA-F) or electrothermal atomization furnace (AA-ETA), inductively coupled plasma-emission spectroscopy (ICP-ES), inductively coupled plasma-mass spectrometry (ICP-MS), and high-performance liquid chromatography-mass spectrometry (LC-MS) are state-of-the-art analytical techniques used to measure metals in biological fluids. They are specific and sensitive and provide the cfinical laboratory with the capability to measure a broad array of metals at clinically significant concentrations. For example, ICP-MS is used to measure several metals simultaneously. Photometric assays are also available but require large volumes of sample and have limited analytical performance. Spot tests are also... 

Most of these measurements are still routinely perfonned with immunoassays because of their simplicity, rapidity and relatively low cost, and despite occasional concerns about their reliability and validity. Serum total T has also been quantitated by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and LC-MS-MS. MS-based methods are often used in research smdies or to confirm immunoassay results. The advantage of chromatography coupled with MS is the high specificity not available with immunoassays because they are susceptible to cross-reactions. An immunoassay might measure a host of structurally related compounds in addition to the target analyte. 

Hypersensitive Measurement of Proteins by Capillary Isoelectric Focusing and Liquid Chromatography-Mass Spectrometry... 

Reliable techniques exist for liquid-liquid extraction of ephedrine from alkaline tissue samples. Gas chromatography-mass spectrometry measurement requires pentafluoropropionic acid derivatization. Blood and tissue measurements have been reported in several ephedrine-related deaths, and in clinical trials with therapeutic doses of the drug (Backer et al., 1997). More recently capillary electrophoresis has been used to separate and identify all 10 stereoisomers of the ephedrine family found in nutritional supplements. Chiral discrimination is effected by using hydroxypropyl-P-cyclodextrin (Flurer et al., 1995). 

An activity-based probe meeting these requirements could, in principle, enable the comparative measurement and molecular identification of all the active members of a given enzyme class present in one or more proteomes. Importantly, these enzyme activity profiles can be read out in a variety of formats including gels [20,25], microarrays [26], liquid chromatography-mass spectrometry (LC-MS) [27], and capillary electrophoresis [28] (Fig. 7.3-3). 

---