Maximizing automation in LC/MS high-throughput analysis and purification

Here, we describe a system for LC/MS-based analysis and purification of compounds aiming at the minimization of manual interference in the overall process. Key elements of the concept are automated identification of the target compounds, automated assignment of optimized preparative gradients for purification of the target compounds, and automated purity assessment of fractions with subsequent pooling of validated product fractions. Additional support is provided by an automated solvent and waste management system. One person can easily process 100-200 compounds on a 150-mg scale per day on that system, while still the maximization of purity and yield after purification is guaranteed. Reduced demands with respect to purity or yield can lead to significantly higher throughput numbers.     

LC/MS and LC/MS/MS determination of protein tryptic digests

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.     

Enhanced performance test mix for high-throughput LC/MS analysis of pharmaceutical compounds

LC/MS is being used for the routine analysis of small molecules in both the discovery and development stages within the pharmaceutical industry. In drug discovery, LC/MS is relied upon to confirm the identity and assess the purity of chemical entities. To ensure the quality of LC/MS analysis, it is important that the LC/MS system is operating within defined performance criteria. Performance monitoring of the system with a standard compound mix offers many advantages over other alternatives, since it monitors the LC/MS system as an integrated unit under the same working conditions as those used for the analysis of samples. It is also a convenient approach, because the test mix can be injected as part of the automated sequence. Use of a test mix for similar purposes has been described previously (Tang, L.; Fitch, W. L.; Alexander, M. S.; Dolan, J. W. Anal. Chem. 2000, 72, 5211-5218). To monitor the performance of ArQule's LC/MS operation (with UV and ELS detection) in greater detail, a set of eight compounds was selected from a collection of 137 commercially available "druglike" compounds. The compounds are generally stable and compliant with the rule-of-five criteria. This enhanced mix has a balanced selection of pKa values and covers the typical range of hydrophobicity and molecular masses of pharmaceutical compounds. Moreover, the selected compounds can generally be ionized using ESI and APCI modes with positive and negative polarity. The test mix can be used under formic acid or ammonium hydroxide conditions and with methanol or acetonitrile as an organic modifier. Performance monitoring with the enhanced mix is demonstrated with respect to ionization and mass measurement, as well as changes in gradient profile, flow rate, buffer pH, and ionic strength.     

Purifying the masses: integrating prepurification quality control, high-throughput LC/MS purification, and compound plating to feed high-throughput screening

In this paper we report using a parallel, four-channel HPLC/MUX/MS purification system, the Purification Factory, to purify thousands of compounds destined for high-throughput screening in a single month. The maximum sample throughput during this 20-workday month was 704 samples/day. Since this purification throughput exceeded the postpurification sample and data handling capabilities provided by commercial solutions, a custom-integrated solution was designed to address these shortcomings. In this paper we detail the key improvements in automation, solvent handling, and sample handling logistics implemented to sustain a mean throughput of 528 samples/day over a multimonth time period.     

Determinations of geniposide using LC/MS/MS methods via forming ammonium and acetate adducts

In this paper, we report method development work to determine geniposide using LC/MS/MS via the formation of positive and negative ion adducts. Geniposide, which has been recognized to have choleretic effects, is the major iridoid glycoside component of Gardenia herbs. To enhance the sensitivity of LC/MS detection of geniposide, a small amount of volatile additives such as ammonium acetate and acetic acid are added into mobile phase solvents to form positive and negative adducts, which can then ionize via electrospray processes. The formation of positive adducts is due to the complexation between geniposide and ammonium ions ([M + NH₄]⁺). The formation of anionic adducts [M + CH₃COO]⁻ is believed to occur via hydrogen bonds bridging acetate ions and glucose groups on the geniposide molecule. Mobile phase solvents containing acetonitrile and aqueous solution (0.2 mM ammonium acetate or 0.1% acetic acid) at the ratio 15: 85 are employed to elute geniposide using C8 reverse phase liquid chromatography columns with electrospray tandem mass spectrometry determinations. Using geniposide standards, the methods are validated at the concentration ranges of 5 to 1000 ng/mL and 20 to 5000 ng/mL using ammonium and acetate adducts respectively. The correlation coefficients of the standard curves are 0.9999 using both ammonium and acetate adducts. The detection limits of using ammonium and acetate adducts are 1 and 5 ng/mL respectively. The measurement accuracy and precision of using ammonium adducts are within 12% and 3% respectively, whereas the accuracy and precision are within 6 and 11% respectively using acetate adducts. When the validated calibration curves of the ammonium adduct of geniposide are used to determine spiked control samples in rat blood dialysates, the determination errors of accuracy and precision are within 12% and 10% respectively.     

Recently developed GC/MS and LC/MS methods for determining NSAIDs in water samples

Pharmaceuticals have become major targets in environmental chemistry due to their presence in aquatic environments (following incomplete removal in wastewater treatment or point-source contaminations), threat to drinking water sources and concern about their possible effects to wildlife and humans. Recently several methods have been developed for the determination of drugs and their metabolites in the lower nanogram per litre range, most of them using solid-phase extraction (SPE) or solid-phase microextraction (SPME), derivatisation and finally gas chromatography mass spectrometry (GC-MS), gas chromatography tandem mass spectrometry (GC-MS/MS) and liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS). Due to the elevated polarity of non-steroidal anti-inflamatory drugs (NSAIDs), analytical techniques based on either liquid chromatography coupled to mass spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS) after a previous derivatisation step are essential. The most advanced aspects of current GC-MS, GC-MS/MS and LC-MS/MS methodologies for NSAID analysis are presented.     

LC/MS and LC/MS/MS based protocol for identification of dyes in historic textiles

Identification of dyes in historic textiles was until recently only based on reversed phase liquid chromatography and diode-array detection (RPLC–DAD). Although in the last years mass spectrometry (MS) is increasingly used as a detection system for liquid chromatography, most applications in the field are directed to identification of the molecular ions or in studies dedicated to degradation products which may be used as markers in RPLC–DAD. In the present work, an analytical protocol for the identification of dyes using RPLC/ESI/MS is presented. Atmospheric pressure electrospray ionization (ESI) was applied, in the negative ion monitoring mode. Both single stage and tandem MS (MS/MS) approaches were considered. An ion trap was used as mass analyzer. Experiments are based on the characterization of standards (natural dyes and/or dyed fibers) with the mass spectrometer sequentially working in the following modes: single MS/full scan, followed by plotting chromatograms through ion extraction (IEC) according to mass/charge ratios corresponding to molecular ions; single MS/selected ion monitoring (SIM) mode; tandem MS/single reaction monitoring (SRM) mode; tandem MS/multiple reactions monitoring (MRM) or product ion scanning modes. A faster chromatographic separation could be applied as MS detection readily balanced the selectivity of the analytical process. In a case study, 11 dyes from 3 biological sources were detected in a 0.5 mg historic sample.     

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.     

Determination of estrogens and progestogens by mass spectrometric techniques (GC/MS, LC/MS and LC/MS/MS)

Steroid sex hormones and related synthetic compounds have been shown to provoke alarming estrogenic effects in aquatic organisms, such as feminization, at very low concentrations (ng/L or pg/L). In this work, different chromatographic techniques, namely, gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), are discussed for the analysis of estrogens, both free and conjugated, and progestogens, and the sensitivities achieved with the various techniques are inter-compared. GC/MS analyses are usually carried out after derivatization of the analytes with bis(trimethylsilyl)trifluoroacetamide (BSTFA). For LC/MS and LC/MS/MS analyses, different instruments, ionization techniques (electrospray (ESI) and atmospheric pressure chemical ionization (APCI)), ionization modes (negative ion (NI) and positive ion (PI)) and monitoring modes (selected ion monitoring (SIM) and selected reaction monitoring (SRM)) are generally employed. Based on sensitivity and selectivity, LC/ESI-MS/MS is generally the method of choice for determination of estrogens in the NI mode and of progestogens in the PI mode (instrumental detection limits (IDLs) 0.1-10 ng/mL). IDLs achieved by LC/ESI-MS in the SIM mode and by LC/ESI-MS/MS in the SRM mode were, in general, comparable, although the selectivity of the latter is significantly higher and essential to avoid false positive determinations in the analysis of real samples. Conclusions and future perspectives are outlined.     

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS(n) experiments

A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 μm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.     

Coupling techniques in LC/MS and SFC/MS

Summary Techniques and applications of analytical instruments combining a chromatographic technique, including liquid chromatography and supercritical fluid chromatography, with mass spectrometry (LC/MS and SFC/MS), that have appeared over the past five years, are reviewed and discussed. It is shown that still many different methods co-exist and have both specific advantages and limitations. SFC/MS appears easier to run for many compounds so far analysed by conventional LC/MS methods. On the other hand, new LC/MS methods that use fast atom bombardment or electrospray ionization have the greater potential for the investigation of polar biopolymers.     

Nanoflow LC/IMS-MS and LC/IMS-CID/MS of protein mixtures

A simple ion trap/ion mobility/time-of-flight (TOF) mass spectrometer has been coupled with nanoflow liquid chromatography to examine the feasibility of analyzing mixtures of intact proteins. In this approach proteins are separated using reversed-phase chromatography. As components elute from the column, they are electrosprayed into the gas phase and separated again in a drift tube prior to being dispersed and analyzed in a TOF mass spectrometer. The mobilities of ions through a buffer gas depend upon their collision cross sections and charge states; separation based on these gas-phase parameters provides a new means of simplifying mass spectra and characterizing mixtures. Additionally it is possible to induce dissociation at the exit of the drift tube and examine the fragmentation patterns of specific protein ion charge states and conformations. The approach is demonstrated by examining a simple three-component mixture containing ubiquitin, cytochrome c, and myoglobin and several larger prepared protein mixtures. The potential of this approach for use in proteomic applications is considered.     

High-throughput quantitative bioanalysis by LC/MS/MS

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.     

LC/MS/MS方法筛查新生儿苯丙酮尿症

苯丙酮尿症 [1～ 4 ] ( PKU )发病原因是患者基因缺陷使肝脏不能合成苯丙氨酸羟化酶而导致体内苯丙氨酸 ( Phe)不能正常代谢为酪氨酸 ( Tyr) ,前者在体内大量堆积并氧化为对人体有害的苯丙酮酸 . PKU是目前筛查范围最广的氨基酸代谢遗传疾病 ,在全世界每年 [5]约有一千万婴儿接受 PKU筛查 ;在我国 ,PKU也是卫生部要求重点筛查的病种 .Chace[5]等在 1 993年报道了 MS/ MS方法筛查新生儿PKU:直接使用 MS/ MS的中性碎片丢失扫描方式检测 Phe和 Tyr,通过氘代内标与待测氨基酸的质谱峰高比来定量 .MS/ MS方法速度快、准确性好、可以…     

Determination of ketamine and amphetamines in hair by LC/MS/MS

A liquid chromatography-tandem mass spectrometry method was developed for the determination of ketamine (with its metabolite norketamine) and some amphetamines (amphetamine, methamphetamine, methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine). This method was developed to determine these compounds in hair and is able to simultaneously quantify all of them in human hair. Hair samples (20 mg) were washed and pulverized, and an extraction with formic acid (0.01%) and ultrasonication for 4 h was used. Deuterated analogs of the analytes were used as internal standards for quantification. Linearity from 0.5 to 25 ng/mg was obtained for both ketamine (and norketamine) and amphetamines with correlation coefficients exceeding 0.99. The limit of detection and the limit of quantification obtained were 0.1 and 0.5 ng/mg, respectively, for ketamine and amphetamines. A total of 25 hair samples from known drug abusers (relating to designer drug consumption or consumption of amphetamines) were examined by this validated method. The results show that the proposed method is suitable for testing these drugs in a single sample of hair. In addition, it is simpler and faster than analysis by conventional methods such as gas chromatography-mass spectrometry, which usually require a more laborious extraction procedure and, in most of cases, an additional derivatization process.