The extensive metabolism and administration of low doses of ethinylestradiol (EE) in preclinical animal species necessitates a sensitive analytical method to quantify the drug at low picogram-per-milliliter concentrations in biological matrixes. A highly sensitive and accurate method based on the derivatization of EE with dansyl chloride coupled with liquid chromatography/tandem mass spectrometry is described. The dansyl derivatization of EE introduced a basic secondary nitrogen into the molecule that was readily ionized in commonly used acidic HPLC mobile phases. The derivative showed an intense protonated molecular ion at m/z 530 under positive turbo ion spray ionization. The collision-induced dissociation of this ion formed a distinctive product at m/z 171, corresponding to the protonated 5-(dimethylamino)naphthalene moiety. The selected reaction monitoring, based on the m/z 530 --> 171 transition, was highly specific for EE, since no background signal was observed from blank plasma obtained from rhesus monkeys. The limit of detection, at a signal-to-noise ratio of 5, was 0.2 fg/mL EE spiked into blank plasma. This allowed for a lower limit of quantitation of 5 pg/mL using a 50-microL plasma sample and 10-microL injection of dansylated derivative into the CTC-PAL Leap autosampler coupled to a Sciex API 4000 mass spectrometer. Using fast-gradient liquid chromatography, the analyte peak eluted at 1.6 min. The validation results showed high accuracy (% bias < 4) and precision (% CV < 7.5) at broad linear dynamic ranges (0.005-20 ng/mL), using deuterated EE as internal standard. Therefore, the facile dansyl derivatization coupled with tandem mass spectral analysis allowed the development of a highly sensitive and specific method for quantitation of trace levels of EE in the plasma of rhesus monkeys dosed orally and intravenously with EE.

Despite recent advances in the application of data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) to the identification of drug metabolites in complex biological matrixes, a prior knowledge of the likely routes of biotransformation of the therapeutic agent of interest greatly facilitates the detection and structural characterization of its metabolites. Thus, prediction of the [M + H]+ m/z values of expected metabolites allows for the construction of user-defined MS(n) protocols that frequently reveal the presence of minor drug metabolites, even in the presence of a vast excess of coeluting endogenous constituents. However, this approach suffers from inherent user bias, as a result of which additional "survey scans" (e.g., precursor ion and constant neutral loss scans) are required to ensure detection of as many drug-related components in the sample as possible. In the present study, a novel approach to this problem has been evaluated, in which knowledge-based predictions of metabolic pathways are first derived from a commercial database, the output from which is used to formulate a list-dependent LC/MS(n) data acquisition protocol. Using indinavir as a model drug, a substructure similarity search on the MDL metabolism database with a similarity index of 60% yielded 188 "hits", pointing to the possible operation of two hydrolytic, two N-dealkylation, three N-glucuronidation, one N-methylation, and several aromatic and aliphatic oxidation pathways. Integration of this information with data-dependent LC/MS(n) analysis using an ion trap mass spectrometer led to the identification of 18 metabolites of indinavir following incubation of the drug with human hepatic postmitochondrial preparations. This result was accomplished with only a single LC/MS(n) run, representing significant savings in instrument use and operator time, and afforded an accurate view of the complex in vitro metabolic profile of this drug.

No abstract

Electron capture dissociation (ECD) and collision-induced dissociation (CID), the two complementary fragmentation techniques, are demonstrated to be effective in the detection and localization of the methionine sulfoxide [Met(O)] residues in peptides using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The presence of Met(O) can be easily recognized in the low-energy CID spectrum showing the characteristic loss of methanesulfenic acid (CH 3 SOH, 64 Da) from the side chain of Met(O). The position of Met(O) can then be localized by ECD which is capable of providing extensive peptide backbone fragmentation without detaching the labile Met(O) side chain. We studied CID and ECD of several Met(O)-containing peptides that included the 44-residue human growth hormone-releasing factor (GRF) and the human atrial natriuretic peptide (ANP). The distinction and complementarity of the two fragmentation techniques were particularly remarkable in their effects on ANP, a disulfide bond-containing peptide. While the predominant fragmentation pathway in CID of ANP was the loss of CH 3 SOH (64 Da) from the molecular ion, ECD of ANP resulted in many sequence-informative products, including those from cleavages within the disulfidebonded cyclic structure, to allow for the direct localization of Met(O) without the typical procedures for disulfide bond reduction followed by ™SH alkylation. (J Am Soc Mass Spectrom 2003, 14, 605-613)

The dissociation of cytochrome c ions (15+ charge state) generated by electrospray ionization has been studied by Fourier transform ion cyclotron resonance mass spectrometry (FTICR) using a sustained off-resonance irradiation/collision-induced dissociation (SORI-CID) technique. Over 95% of the fragment ions can be accurately assigned (to better than 10 ppm), yielding information on the primary sequences of the various proteins. Up to four stages of mass spectrometry (MS4) have been achieved without the need for quadrupole excitation/collisional cooling of the product ions. The subtle structural differences among the cytochrome c variants (from bovine, tuna, rabbit, and horse) are clearly reflected in their fragmentation patterns: replacing 3 out of 104 residues of the cytochrome c is shown to dramatically change the dissociation pattern. Of particular importance are a variety of results indicating that the dissociation of the cytochrome c's is influenced by higher-order structure and charge location, in addition to the primary structure (i.e., sequence). No fragmentation is observed in the region between residues 10-20 and little dissociation between residues 70-90. This is most likely due to the interactions of the heme group with the polypeptide chain, and such a heme "footprinting" pattern is analogous to the protein conformation in solution. These studies demonstrate that electrospray ionization-FTICR using SORI-CID can be a useful tool to probe not only the small differences in the primary sequences of proteins but also suggest the potential for probing their higher-order structures and yielding information not readily available from H/D exchange or circular dichoism studies.

A new type of quadrupole linear ion trap mass spectrometer, Q TRAP TM LC/MS/MS system (Q TRAP TM ), was evaluated for its performance in two studies: firstly, the in vitro metabolism of gemfibrozil in human liver microsomes, and, secondly, the quantification of propranolol in rat plasma.With the built-in information-dependent-acquisition (IDA) software, the instrument utilizes full scan MS in the ion trap mode and/or constant neutral loss scans as survey scans to trigger product ion scan (MS 2 ) and MS 3 experiments to obtain structural information of drug metabolites 'on-thefly'. Using this approach, five metabolites of gemfibrozil were detected in a single injection. This instrument combines some of the unique features of a triple quadrupole mass spectrometer, such as constant neutral loss scan, precursor ion scan and multiple reaction monitoring (MRM), together with the capability of a three-dimensional ion trap. Therefore, it becomes a powerful instrument for metabolite identification. The fast duty cycle in the ion trap mode allows the use of full product ion scan for quantification. For the quantification of propranolol, both MRM mode and full product ion scan in the ion trap mode were employed. Similar sensitivity, reproducibility and linearity values were established using these two approaches. The use of the product ion scan mode for quantification provided a convenient tool in selecting transitions for improving selectivity during the method development stage. Copyright # 2003 John Wiley & Sons, Ltd.Liquid chromatography/tandem mass spectrometry (LC/ MS/MS) has been widely applied in both qualitative and quantitative analyses of drug candidates and their metabolites in biological matrices, in support of drug discovery and development. 1-3 Commonly used tandem mass spectrometers include triple stage quadrupole (tandem-in-space), three-dimensional (3D) ion trap (tandem-in-time), and quadrupole time-of-flight (Q-TOF) mass spectrometers. Threedimensional ion trap mass spectrometers are widely used for conducting consecutive product ion scans (MS n ) in a data-dependent fashion that provides useful information for structural elucidation. Triple quadrupole mass spectrometers, on the other hand, can be used to conduct tandem mass scan experiments in various modes including product ion scan (MS 2 ), precursor ion scan, constant neutral loss scan, and multiple reaction monitoring (MRM). The constant neutral loss and precursor ion scan experiments on a triple quadrupole serve as a convenient tool for detecting unknown metabolites in complex biological matrices, especially for phase II drug metabolites, such as acyl glucuronide (loss of 176 Da) and sulfate (loss of 80 Da) conjugates. Thus far, MRM is the most sensitive and selective detection method for confirming trace levels of circulating metabolites in plasma, 4 for example, as in human ADME studies, as well as in animal organs, and it is the method of choice for quantifying drugs and their metabolites in biological fluids. Nevertheless, both instruments have...

A Fourier transform ion cyclotron resonance (FTICR) mass spectrometer has been used to trap individual multiply charged ions of several high molecular weight polymers, including poly(ethy1ene oxide), sodium poly(styrene sulfonate), and the protein bovine serum albumin. Detection of these ions is performed with the nondestructive method distinctive of FTICR, which also allows remeasurement of the same ion or ion population over several hours. For the determination of the charge states (and hence the masses) of individual ions, a new scheme was developed on the basis of the observation of the stepwise m/z shifts that result from charge exchange reactions or adduction of a substance of known mass. A novel technique for mass determination of individual ions has been made possible with theobservation of cyclotron frequency shifts during the time-domain acquisition period. This time-resolved ion correlation (TRIC) technique allows reactant and product ions to be correlated with confidence and provides the basis for simultaneously studying a moderate number of ions. In this work, a range of observations related to the detection and measurement of individual ions is presented, as are examples of mass determinations of individual ions performed by utilizing the TRIC technique. Results are presented that show the measurement of poly(ethy1ene glycol) (PEG) individual ions of more than 5 MDa with more than 2500 net charges and measurement of ions as small as albumin (66 kDa) with as few as 30 charges. Other results illustrate that unexpected behavior can be observed for individual ions that would not be apparent in large ion populations.

No abstract