High-performance liquid precursor analysis

Tian, Q. et al., Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring, J. Chromatogr. A, 1091, 72, 2005. 

Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

High-performance liquid chromatography (HPLC), followed by GC/MS, has been used to fractionate and then quantitate the aliphatic and aromatic hydrocarbons present in liquid fuel precursors in order to determine the fuel potential of the compounds. Kerosene had the advantage of not requiring any sample preparation. Other light fuel oils may require the use of methylene chloride as a solvent prior to HPLC analysis (Lamey et al. 1991). The sensitivity, precision, and recovery of this method were not reported. 

The stationary phase may be a solid or liquid on a solid support. The mechanisms responsible for distribution between phases include surface absorption, ion exchange, relative solubilities and steric affects . High performance liquid chromatography is a useful method for quinolizidine alkaloid analysis, especially when pure standards are available". This method was recently used for alkaloid metabolite extraction and analysis . A simple reversed-phase liquid chromatographic method has been developed for the simultaneous quantitation of four anticancerous alkaloids vincristine, vinblastine, and their precursors catharanthine and vindoline using a specific HPLC column . 

Superior sensitivity, efficiency, and specificity have made high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), the predominant analytical technique for characterization and quantitative analysis of metabolites (Kostiainen et al., 2003 Ma et al., 2006 Prakash et al., 2007). Ion trap, triple-quadrupole, and quadmpole time-of-flight (Q-TOF) mass spectrometers are routinely used to profile and characterize metabolites in plasma and excreta (Ma et al., 2006). The combination of scan types and features available on mass spectrometers of different design (product ion, MS", neutral loss, precursor ion scans, accurate mass measurements) allows identification and characterization of putative and unexpected metabolites with or without little prior knowledge of biotransformation pathways of a given dmg molecule. 

Squalene is a precursor for steroid biosynthesis and may have cardioprotective and cancer-preventative properties. Though nuts are known to contain squalene, the limited literature available indicates that the pine nut contains a relatively low amount of squalene compared to other nuts [8]. Reverse-phase high-performance liquid chromatography (HPLC) analysis showed that pine nut oil had a squalene content of about 40mg/100g oil, which is approximately 23mg/100 nuts. Thus, they would not be a likely choice for a squalene-based nutraceutical however, this study did not report the species of pine nut measured, and variability between species of other compounds of interest may indicate that a different species could provide higher squalene content. 

---