A sensitive high-performance liquid chromatographic assay was developed using chloride attachment negative chemical ionization mass spectrometry for detection of glyceryl esters and ceramides, and positive chemical ionization mass spectrometry for detection of free cholesterol and cholesteryl esters in minimal quantities of plasma. The novel technique was validated by high temperature gas-liquid chromatography with flame ionization detection. Sample preparation was achieved by phospholipase C digestion of whole plasma, total lipid extraction and derivatization of any free carboxyl and hydroxyl groups by trimethyl- or tert-butyldimethyl-chlorosilane. The lipids were separated by reverse phase HPLC with 20-90% propionitrile in acetonitrile containing 1% dichloromethane, which served as the reagent and the source of chloride. Negative chemical ionization with chloride attachment is estimated to provide about 100 times higher response for the triacylglycerols and the trimethylsilyl or tert-butyldimethylsilyl ethers of diacylglycerols, and about 500 times higher response for the trimethylsilyl or tert-butyldimethylsilyl ethers of ceramides than positive chemical ionization mass spectrometry. Determination of the full negative chemical ionization mass spectra showed that each glycerolipid and ceramide species yielded a single ionic species corresponding to the chloride-attachment product of the parent ion. The cholesteryl esters and ethers failed to attach chloride and remained undetected by negative chemical ionization. However, the cholesteryl esters and ethers gave a high response for the steroid nucleus in positive chemical ionization mass spectrometry. Chloride attachment negative chemical ionization mass spectrometry is suitable for the unequivocal identification of plasma glycerolipids and ceramides in high-performance liquid chromatography and for the quantitation of molecular species in any unresolved peaks following appropriate calibration of the instrument response.