Distribution measurement of amphetamine-type stimulants in organs using micropulverized extraction and liquid chromatography/tandem mass spectrometry to complement drug distribution using mass spectrometry imaging

Amphetamine-type stimulants (ATS) such as methamphetamine are widely abused and can cause toxic effects in the body. In this study, a simple and accurate analytical method for distribution measurement of drugs in organs was developed to visualize localization of ATS in organs and to complement drug distribution by mass spectrometry imaging (MSI). The brain, liver and kidney from rats to which ATS had been administered were segmented into blocks of 2×2×2 mm3 at -30°C. Each organ block was micropulverized with a stainless-steel bullet at -80°C. The concentrations of drugs in each block were measured by liquid chromatography/tandem mass spectrometry. The three-dimensional distribution of drugs in a whole organ was expressed using color gradation of drug concentration after reconstruction of all blocks to the original locations. The distribution was also compared with that obtained by MSI. This method enabled measurement of drug distribution in organs with simple and clean procedures and accurate quantification unlike autoradiography and MSI. The methamphetamine concentrations were different between parts in an organ, particularly in the kidney. This method could be applicable to the measurement of the distribution of compounds in various solid samples and could be used as a complementary method for the measurement of the distribution of compounds by MSI.     

Quantitative analysis of erythropoietin in human plasma by tandem mass spectrometry

The extended use of protein drugs in therapeutics has created the need for their quantification in human plasma. A methodology using the therapeutic protein itself as internal standard for quantitative analysis by multiple reaction monitoring (MRM) has been designed and applied to epoetin beta, a recombinant human erythropoietin (rhEPO). After depletion of major proteins, plasma samples were desalted and enriched in rhEPO by reversed phase liquid chromatography prior to tryptic cleavage. Differential isotopic labeling of peptides was performed by derivatization with 2-methoxy-4,5-dehydro-imidazole. A light version (four hydrogen atoms) of this reagent was used for plasma peptides. Tryptic peptides obtained from pure rhEPO were derivatized with a heavy version (four deuterium atoms) of the same reagent and used as internal standards. Two rhEPO tryptic peptides with three MRM transitions per peptide were selected for quantification. This strategy provided a quantification limit close to 50 amol of epoetin beta per microliter of plasma (equivalent to 1.7 ng/mL), i.e., well below the expected therapeutic concentrations in plasma (around 100–500 amol/μL).     

Quantitative analysis of microstructures by secondary ion mass spectrometry.

The focus of this review is on trace-element quantitation of microstructures in solids. This review is aimed at the nonspecialist who wants to know how secondary ion mass spectrometry (SIMS) quantitation is achieved. Despite 35 years of SIMS research and applications, SIMS quantitation remains a fundamentally empirical enterprise and is based on standards. The most used standards are "bulk standards"-solids with a homogeneous distribution of a trace element-and ion-implanted solids. The SIMS systematics of bulk standards and ion-implanted solids are reviewed.     

Determination of pharmaceutical compounds in skin by imaging matrix-assisted laser desorption/ionisation mass spectrometry

Matrix-assisted laser desorption/ionisation (MALDI) quadrupole time-of-flight mass spectrometry (Q-TOFMS) has been used to detect and image the distribution of a xenobiotic substance in skin. Porcine epidermal tissue was treated with 'Nizoral', a medicated shampoo containing ketoconazole (+/-)-1-acetyl-4-[p-[[(2R,4S)-2-(2,4-dichlorophenyl)-2-(imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) as active ingredient. Following incubation for 1 h at 37 degrees C all excess formulation was washed from the surface. A cross-section of the drug-treated tissue was then blotted onto a cellulose membrane, precoated in matrix (alpha-cyano-4-hydroxycinnamic acid (CHCA)), by airspray deposition. In separate experiments the tissue surface was treated with Nizoral within a triangular former, and subsequently blotted onto a matrix-coated membrane. Sample membranes were then mounted into the recess of specialised MALDI targets with adhesive tape. All samples were analysed by MALDI-TOFMS using an Applied Biosystem 'Q-star Pulsar i' hybrid Q-TOF mass spectrometer fitted with an orthagonal MALDI ion source and imaging software. Detection of the protonated molecule was readily achievable by this technique. Treatment of the tissue within a template gave rise to images depicting the expected distribution of the drug, demonstrating that this technique is capable of producing spatially useful data. Ion images demonstrating the permeation of the applied compound into the skin were achieved by imaging a cross-sectional imprint of treated tissue. A calibration graph for the determination of ketoconazole was prepared using the sodium adduct of the matrix ion as an internal standard. This enabled construction of a quantitative profile of drug in skin. Conventional haematoxylin and eosin staining and microscopy methods were employed to obtain a histological image of the porcine epidermal tissue. Superimposing the mass spectrometric and histological images appeared to indicate drug permeation into the dermal tissue layer.     

Quantitative analysis by thin-layer chromatography with secondary ion mass spectrometry

Summary A direct combination of thin-layer chromatography with secondary ion mass spectrometry (TLC/SIMS) provides a method for the quantitative analysis of thermally unstable compounds or compounds of low volatility such as nicergoline. The method is very simple and has excellent precision. The analysis was performed by using an aluminium TLC plate and a mixture of methylene chloride, acetone, and distilled water as a developing solvent. After development the portion of the plate with the nicergoline and the internal standard spots was cut off the TLC plate, and was attached to the SIMS holder directly. The amount of nicergoline was determined from the ratio of the fragment ion intensity of the nicergoline to the internal standard. The calibration curve was linear, and the detection limit was 10 ng at a signal-to-noise ratio of 5. This method should be considered for application to the determination of drugs in biological samples and also for the determination of possible impurities and decomposition products in drugs.     

Fragmentation study of hexanitrostilbene by ion trap multiple mass spectrometry and analysis by liquid chromatography/mass spectrometry

The fragmentation pathways of three explosive compounds with similar structures, hexanitrostilbene (HNS), cyclotrimethylene trinitramine (RDX), and 2,4,6-trinitrotoluene (TNT), have been investigated by multiple mass spectrometry (MSn, n = 1, 2, 3) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. The electron capture mechanism for these compounds in negative ion APCI and ESI mode differs from the usual negative ion mechanism, deprotonation or addition of other species. This was shown for HNS and TNT, which both gave a [M]- anion but not a [M-H]- ion in APCI, and the [M]- anion of HNS was observed in ESI. The quantitative analysis of HNS was performed by liquid chromatography (LC)/ESI-MS, and the results obtained by the internal standard (ISTD) method were compared with those from the external standard (ESTD) method, demonstrating that both quantitation approaches are useful, with good sensitivity, reproducibility and linearity, and ESTD is preferable in routine applications.     

Quantitative analysis of multiple components based on liquid chromatography with mass spectrometry in full scan mode

Although liquid chromatography with mass spectrometry in full scan mode can obtain all the signals simultaneously in a large range and low cost, it is rarely used in quantitative analysis due to several problems such as chromatographic drifts and peak overlap. In this paper, we propose a Tchebichef moment method for the simultaneous quantitative analysis of three active compounds in Qingrejiedu oral liquid based on three‐dimensional spectra in full scan mode of liquid chromatography with mass spectrometry. After the Tchebichef moments were calculated directly from the spectra, the quantitative linear models for three active compounds were established by stepwise regression. All the correlation coefficients were more than 0.9978. The limits of detection and limits of quantitation were less than 0.11 and 0.49 μg/mL, respectively. The intra‐ and interday precisions were less than 6.54 and 9.47%, while the recovery ranged from 102.56 to 112.15%. Owing to the advantages of multi‐resolution and inherent invariance properties, Tchebichef moments could provide favorable results even in the situation of peaks shifting and overlapping, unknown interferences and noise signals, so it could be applied to the analysis of three‐dimensional spectra in full scan mode of liquid chromatography with mass spectrometry.     

Quantitative analysis of crude plasma extracts by ion trap tandem mass spectrometry

The ion trap mass spectrometer is a tandem-in-time instrument that has promise as an extremely sensitive device for practical tandem mass spectrometry assays. An approach for the quantitative analysis of unknown drug levels in crude extracts, using combined capillary gas chromatography and the ion trap mass spectrometer in the tandem mode, is described. One-gram plasma samples were spiked with an anti-inflammatory drug at levels of 1–100 ng, and with 50 ng of a chemical analog internal standard. Crude extracts of the plasma samples are analyzed by using scan functions that utilize combined radiofrequency (rf) and de voltages. The need for combined rf- and de-voltage sequences for analysis of such extracts is demonstrated by comparison to attempted analyses using only rf voltages. Limitations of the method are: (1) the need for accurate calibration of ionization times to obtain linear calibration lines, and (2) the lack of automatic gain control for scans using combined rf and dc voltages to control and optimize parent ion populations and to allow a simpler analysis of “unknowns. ”     

Options for veterinary drug analysis using mass spectrometry

Several classes of chemical compounds, exhibiting many different chemical properties, are classified under the generic term of “veterinary drugs”, among which are the antimicrobial medicines such as antibiotics or dyes, and drugs exhibiting growth promoting properties like steroids, β-agonist compounds, thyrostats or growth hormones. For food safety purposes, the resort to these substances in animal breeding has been submitted to strict regulation within the European Union for more than 15 years. Systems of control have therefore been set up within the same period of time to ensure compliance with the regulation. The current strategy relies on targeted analytical approaches focusing on the detection of residues of the administered compounds or their metabolites in different kinds of feed, food or biological matrices. If screening methods, which provide rapid discrimination between compliant and suspect samples, may be based on several techniques such as immunoassays or mass spectrometry, confirmatory methods mainly rely on the latter, which provides adequate specificity and sensitivity for unambiguous identification of the target analytes in biological matrices at trace level. The present article reviews the main mass spectrometric strategies, from the very first, nonetheless still efficient, single MS and multidimensional and high-resolution MS through to advanced isotope ratio MS. Several applications in the field of residue analysis illustrate each of these approaches and focus on the balance between issues related to the compounds of interest (chemistry, matrix, concentration, …) and the large offer of mass spectrometric-related technical possibilities, from the choice of the ionization conditions (EI, NCI, PCI, reagent gases, ESI+, ESI?), to the mass analyzers (single quadrupole, triple quadrupole, ion traps, time-of-flight, magnetic sectors, isotope ratio mass spectrometer) and corresponding acquisition modes (full scan, LR–SIM, HR–SIM, SRM, precursor scan, …). All the displayed strategies, from the importance of sample preparation to MS analysis to potential derivatization steps and chromatographic separation parameters are discussed in that context. Besides the advantages of each strategy, main issues associated to such MS approaches are commented with an emphasis not only on such critical points as ion suppression and resolution, but also on the adequacy of the current regulation regarding the evolution of the technology. Finally, future trends which may lead to strong and positive impacts in the field of residue analysis are presented, including latest developments and improvements in chromatography or software dedicated to signal acquisition and data analysis.     

Visualization of acetylcholine distribution in central nervous system tissue sections by tandem imaging mass spectrometry

Metabolite distribution imaging via imaging mass spectrometry (IMS) is an increasingly utilized tool in the field of neurochemistry. As most previous IMS studies analyzed the relative abundances of larger metabolite species, it is important to expand its application to smaller molecules, such as neurotransmitters. This study aimed to develop an IMS application to visualize neurotransmitter distribution in central nervous system tissue sections. Here, we raise two technical problems that must be resolved to achieve neurotransmitter imaging: (1) the lower concentrations of bioactive molecules, compared with those of membrane lipids, require higher sensitivity and/or signal-to-noise (S/N) ratios in signal detection, and (2) the molecular turnover of the neurotransmitters is rapid; thus, tissue preparation procedures should be performed carefully to minimize postmortem changes. We first evaluated intrinsic sensitivity and matrix interference using Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry (MS) to detect six neurotransmitters and chose acetylcholine (ACh) as a model for study. Next, we examined both single MS imaging and MS/MS imaging for ACh and found that via an ion transition from m/z 146 to m/z 87 in MS/MS imaging, ACh could be visualized with a high S/N ratio. Furthermore, we found that in situ freezing method of brain samples improved IMS data quality in terms of the number of effective pixels and the image contrast (i.e., the sensitivity and dynamic range). Therefore, by addressing the aforementioned problems, we demonstrated the tissue distribution of ACh, the most suitable molecular specimen for positive ion detection by IMS, to reveal its localization in central nervous system tissues.     

Identification of the 'wrong' active pharmaceutical ingredient in a counterfeit Halfan drug product using accurate mass electrospray ionisation mass spectrometry,accurate mass tandem mass spectrometry and liquid chromatography/mass spectrometry

Methodology is presented for identifying an unknown active (pharmaceutical) ingredient (AI) in a counterfeit drug product. A range of mass spectrometric techniques, i.e., accurate mass mass spectrometry, tandem mass spectrometry (MS/MS) and liquid chromatography/mass spectrometry (LC/MS), has been employed to determine the AI in a counterfeit Halfan suspension, an antimalarial drug. In particular, use of LockSpray accurate mass MS/MS allowed identification of parts of the molecule from fragments, hence limiting the number of possible elemental compositions for the nominal mass of 278 found for the AI in the counterfeit product. The analysis of the isotope pattern observed for the protonated molecule further reduced the number of possible elemental compositions. A literature search for readily commercially available compounds of molecular formula C(12)H(14)N(4)O(2)S suggested that the AI was either sulfamethazine or sulfisomidine. An LC/MS separation of those two compounds and reference MS/MS spectra obtained for sulfamethazine and sulfisomidine led to the conclusion that the AI in the counterfeit Halfan suspension is sulfamethazine, which is an antibacterial agent.     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Quantitative analysis of ezetimibe in human plasma by gas chromatography‐mass spectrometry

A new, specific and sensitive GC‐MS method with electron impact ionization technique was developed for quantitative analysis of ezetimibe (EZE) in human plasma. Prior to GC analysis, EZE was derivatized with N‐methyl‐N‐trimethylsilyl‐trifluoroacetamide (MSTFA), which is a trimethyl silylating reagent. The derivatization reaction was optimized and parameters such as catalyst, derivatization time, temperature, solvent and the volume of silylating reagent were investigated. Trimethylsilyl ether derivative of EZE was determined in selected ion monitoring (SIM, mass‐to‐charge ratio (m/z): 326) mode. The method was validated with respect to LOD and LOQ, precision, accuracy, linearity, specificity, stability, and recovery. The LOQ and LOD were found as 15 and 10 ng/mL, respectively. The linearity of the method ranged from 15 to 250 ng/mL. The correlation coefficient of the calibration curve was 0.9977 ± 0.0004 (± S.E.M.). The intra‐ and inter‐day precisions (RSD) were less than 6% and accuracies (bias) for intra‐ and inter‐day accuracy were found between –4.04 and 9.71% at four different concentration levels (15, 40, 100, 250 ng/mL). The proposed method was successfully applied to real human plasma samples for determination of total EZE.     

Glycopeptide analysis by mass spectrometry

Glycosylation is one of the most important post-translational modifications found in nature. Identifying and characterizing glycans is an important step in correlating glycosylation structure to the glycan's function, both in normal glycoproteins and those that are modified in a disease state. Glycans on a protein can be characterized by a variety of methods. This review focuses on the mass spectral analysis of glycopeptides, after subjecting the glycoprotein to proteolysis. This analytical approach is useful in characterizing glycan heterogeneity and correlating glycan compositions to their attachment sites on the protein. The information obtained from this approach can serve as the foundation for understanding how glycan compositions affect protein function, in both normal and aberrant glycoproteins.