Defining the biologic roles of human milk oligosaccharides (HMOS) requires an efficient, simple, reliable, and robust analytical method for simultaneous quantification of oligosaccharide profiles from multiple samples. The HMOS fraction of milk is a complex mixture of polar, highly branched, isomeric structures that contain no intrinsic facile chromophore, making their resolution and quantification challenging. A liquid chromatography-mass spectrometry (LC-MS) method was devised to resolve and quantify 11 major neutral oligosaccharides of human milk simultaneously. Crude HMOS fractions are reduced, resolved by porous graphitic carbon HPLC with a water/acetonitrile gradient, detected by mass spectrometric specific ion monitoring, and quantified. The HPLC separates isomers of identical molecular weights allowing 11 peaks to be fully resolved and quantified by monitoring mass to charge (m/z) ratios of the deprotonated negative ions. The standard curves for each of the 11 oligosaccharides is linear from 0.078 or 0.156 to 20 μg/mL (R2 > 0.998). Precision (CV) ranges from 1% to 9%. Accuracy is from 86% to 104%. This analytical technique provides sensitive, precise, accurate quantification for each of the 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation.

Saikosaponins are bioactive oleanane saponins derived from the Chinese medicinal herb Radix bupleuri ("chaihu" in Chinese). An LC-MS/MS-based method has been developed for characterization and quantification of 15 saikosaponin derivatives (saikosaponin a, saikosaponin b(1), saikosaponin g, saikogenin A, saikogenin H, saikosaponin c, saikosaponin h, saikosaponin i, prosaikogenin C(2), prosaikogenin B(2), saikogenin C, saikogenin B, saikosaponin d, saikosaponin b(2), and saikogenin D) in one chromatographic run. Optimization of the ionization process was performed with electrospray and atmospheric pressure chemical ionization techniques in both positive and negative ion modes. Negative ion ESI was adopted for generation of the precursor deprotonated molecules to achieve the best ionization sensitivity for the analytes. In addition, the most abundant fragment ion was chosen for each analyte to give the best CID sensitivity. Because some of the saponin derivatives are isomeric, complete resolution for the whole analytes was achieved both chromatographically and mass spectroscopically. Furthermore, optimal internal standard was successfully discovered for determination of the analytes by making use of a combinatorial chemistry approach. Good linearity over the range approximately 1.65 or 4.98 to 1200 ng/mL for the analytes was observed. The intraday accuracy and precision at nominal low, intermediate, and high concentration varied between 0.8 and 11.8% and between 80 and 116%, respectively, whereas those for interday assay were between 1.1 and 15.5% and between 86 and 119%, respectively. The lower limits of quantitation for the test compounds were approximately 16.5 to 49.4 pg on-column. The new method offered higher sensitivity and greater specificity than previously reported LC methods. After the validation, the applicability of the method for determination of these chemicals present in a variety of crude chaihu roots and in different brands of the Chinese multiherb remedy Xiaochaihu-tang (or Shosaiko-to) extract granules has been demonstrated. The sensitivity and specificity of the technique will be the basis of a method for the accurate quantification of the saikosaponin derivatives in biomatrixes.

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange HPLC, reverse or normal phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection, and, in our laboratory, by CE with detection at 205 nm. The novel method described herein uses a running buffer of aqueous 200 mM NaH 2 PO 4 at pH 7.05 containing 100 mM SDS made 45% (v/v) with methanol to baseline resolve five oligosaccharides, and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-minute run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants. Keywordshuman milk oligosaccharides; sialyloligosaccharides; capillary electrophoresis INTRODUCTORY STATEMENTHuman milk contains a large number of compounds that provide nutritional support and contribute toward defense of the newborn. Human milk oligosaccharides (HMOS), a principal component of human milk, are found in concentrations of 6 to 12 g/L, and these oligosaccharides vary in size, structure, and abundance [1]. The oligosaccharides of milks from other species vary widely, and the composition of oligosaccharides from human milk is unique [2].HMOS play a potentially protective role to nursing infants. Breastfed infants have a different microbiota than infants fed artificially, and human milk glycans are thought to act as prebiotics that promote the growth of Bifidobacterium bifidus [3]. Breast-fed infants have fewer or less severe gastrointestinal and respiratory infections during the first year of life than formula-fed