Using MS to quantify drug and lipid concentrations
Mass spectrometry is often the analytical method of choice for detecting, characterizing, and quantifying clinical and biochemical analytes of interest, including drugs of abuse confirmation by isotope dilution gas chromatography-quadrupole-MS (GC/MS); therapeutic drug monitoring and complex lipid quantitation by liquid chromatography-electrospray-tandem MS (LC/ESI/MS/MS) on a triple quadrupole MS; protein identification and genotyping by matrix assisted laser desorption ionization time-of-flight MS (MALDI/TOF/MS); general unknown drug screening by LC/ESI/MS/MS on QTRAP, QTOF, and triple quadrupoles; and accurate mass measurements in drug identification.
An example of drugs of abuse confirmation by isotope dilution GC/MS is the quantitation of urine content of the cocaine metabolite benzoylecgonine, as illustrated in Figures 22-24 (38). Cocaine is a carboxylic acid ester that is rapidly hydrolyzed in vivo to the free acid benzoylecgonine (BEG) (Figure 22), and detection of BEG is used to monitor cocaine abuse.
BEG itself has poor vapor phase properties because of the polar, ionizable carboxylate moiety, and it is therefore converted to a carboxylic acid ester, in this example the pentafluoropropyl (PFP) ester, before GC/MS analysis (Figure 22). The molecular ion of PFP-BEG is m/z 421, and two characteristic fragment ions of m/z 316 and m/z 300 that arise from cleavages about an oxygen atom of the ester (Figure 23). A ring-labeled [2H3]-BEG internal standard is added before extraction, processed along with the target analyte, and also derivatized (d3-PFP-BEG). The ions in the mass spectrum of d3-PFP-BEG analogous to those in the spectrum of PFP-BEG are m/z 424, 319, and 303, and in each case retain all three [2H] atoms. GC/MS is then performed with selected ion monitoring of the ion pairs m/z 316 and 319, m/z 300 and 303, and m/z 421 and 424. Qualitative confirmation is achieved when these ions exhibit an appropriate relative abundance ratio at the correct GC retention time, and quantitation is achieved by comparing the intensity of the ion current from the internal standard and the target analyte (Figure 24).
An example of therapeutic drug level monitoring by LC/ESI/MS/MS on a triple quadrupole MS is the measurement of the immunosuppressant Tacrolimus by selected reaction monitoring (39). Tacrolimus has no ionizable moieties in its structure (Figure 25), but when ammonium acetate is included in the infusion buffer, Tacrolimus complexes with ammonium. This adduct enters the gas phase upon ESI, and its tandem mass spectrum contains [M + NH4]+ (m/z 821.4) and ions reflecting neutral loss of (NH3 + H2O) (m/z 786.4) and of (NH3 + 2H2O) (m/z 768.4) (Figure 26). The tandem mass spectrum of a structural analog used as an internal standard contains analogous ions [M + NH4]+ (m/z 809.5) and ions reflecting neutral loss of (NH3 + H2O) (m/z 774.5) and of (NH3 + 2H2O) (m/z 756.5) (Figure 26). When LC/ESI/MS/MS is performed on a triple quadrupole MS, the reactions 821.5 –> 768.4 and 809.5 –> 756.4 identify Tacrolimus and the internal standard, respectively, at the appropriate LC retention time, and the relative abundance of the ion currents of the two reactions reflects the quantity of Tacrolimus in the sample (Figures 26-27).
Constant neutral loss scanning on a triple quadrupole MS to quantitate ceramide is illustrated in Figures 28-30 (40). Ceramide is a fatty acid amide of the long-chain base sphingosine and is an important signaling molecule in apoptosis (programmed cell death) in many cases. One example is apoptosis of insulin-secreting b-cells induced by depletion of internal Ca2+ stores after treatment with thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase. Ceramide has no ionizable moieties, but it complexes with Li+ in salts added to the infusion buffer. The adduct ion enters the gas phase upon ESI. Cell extracts contain ceramide species with various fatty acid substituents, and an internal standard that does not occur naturally with an octanoic acid substituent (8:0-CM) can be added. Figure 28A illustrates the profile of ceramide species in a lipid extract from insulinoma cells to which 8:0-CM had been added. The 8:0-CM [M + Li]+ ion is m/z 432, and the [M + Li]+ ions of endogenous ceramide species with different fatty acid substituents are m/z 656.7 (24:0-CM), 654.4 (24:1-CM), 628.6 (22:0-CM), 572.5 (18:0-CM), and 544.5 (16:0-CM). Figure 28B is the tandem spectrum from CID of m/z 654.6, which is [M + Li]+ of 24:1-CM. The prominent fragment ion of m/z 606 reflects sequential neutral losses of water and formaldehyde (Figure 29), and the net loss of 48 m/z units is a common feature of the tandem spectra of Li+ adducts of all ceramide species. A constant neutral loss of m/z 48 using ESI/MS/MS scanning on a triple quadrupole-MS can thus be used to display the ceramide species in a biological mixture, and each species can be quantitated relativeto the 8:0-CM internal standard (Figure 30), which compares ceramide content of control insulinoma cells to that of thapsigargin-treated cells. Treated cells exhibit a higher abundance of endogenous species (e.g., m/z 546 for 16:0-CM) relative to the internal standard (m/z 432; 8:0-CM) than control cells, reflecting a thapsigargin-induced rise in endogenous ceramide concentration. The advantage of constant neutral loss scans over simply monitoring total ion current is that MS/MS scanning eliminates interference from extraneous substances in biological extracts and greatly increases both measurement specificity and sensitivity by reducing noise level.