Splitless Injection Research Articles

Large-volume injection gas chromatography-mass spectrometry for automated broad-spectrum drug screening in horse urine.

A rapid, sensitive, and rugged method for detecting drugs and drug metabolites in extracts of horse urine is described. The use of large-volume injection (LVI) gas chromatography-mass spectrometry (GC-MS) for analysis of horse urine extracts allowed automation of the derivatization procedure and reduction of the sample volume from 5 mL to 1 mL of urine. An autosampler and temperature-programmable inlet were used to automatically dissolve the sample extract and form trimethylsilyl derivatives of over 200 analytes. The suitability of this procedure for routine GC-MS detection of approximately 80 basic analytes in extracts of racehorse urine was investigated. The formation of derivatives using LVI with in-liner derivatization was compared to a manual procedure involving the dissolution of sample extracts in N,O-bis(trimethylsily)trifluoroacetamide, heating the resulting mixture, and injecting 1 or 2 microL of the mixture through a splitless injector into the GC-MS instrument. In all cases, the in-liner derivatization reactions were found to be as complete as conventional heating block procedures. Ruggedness testing of the method demonstrated that peak resolution, shape, and area were maintained through 40 consecutive injections of sample extracts. No evidence of the accumulation of interfering substances was observed. The limits of detection using LVI GC-MS for routine screening of basic drugs in urine were generally in the range of 5-25 ng/mL. The method is currently being used to detect basic analytes in horse urine extracts with a throughput of approximately 50 urine sample extracts per instrument per day.

Solid phase micro extraction coupled with on-column GC/ECD for the post-blast analysis of organic explosives

Gas chromatographic analysis with electron capture detection is very sensitive to post-blast residues and useful for the determination of organic explosive molecules. But many compounds extracted from the matrices may interfer with the explosives. Using SPME, most interfering compounds are eliminated so the identification is easier. Another advantage of the technique is a low limit of detection. In this study, four different SPME fibers were tested to analyze the most common encountered organic explosives including nitro aromatics, nitramines and nitro-esters. Different parameters were tested (desorption time, agitation, …) and a special device has been created to optimize the agitation. Direct desorption effect of the SPME fiber on the column compared to normal split–splitless injection is shown. In this way, the degradation of the most sensitive molecules is decreased. An application to a real case is also described in this paper.

Advanced GC/ICP-MS design for high-boiling analyte speciation and large volume solvent injection

A home made multifunctionary interface has been developed to connect a capillary gas chromatograph (GC) and an inductively coupled plasma mass spectrometer (ICP-MS). It can analyze high boiling analytes with a boiling point as high as that of C26 n-paraffin (412 °C) at only 140 °C temperature in the transfer line. The design performs splitless large volume solvent injection to analyze sensitively very low and high boiling analytes in one run. The thin and flexible transfer line is heated by a combination of hot argon mixed with the GC effluent before passing the transfer line using a T-joint in the GC oven, and by electrical resistance heating. The flow through the transferline can be reversed to eliminate the solvent peak and to prevent graphite deposition onto the ICP-MS cones after large volume splittless injection. The detection limits for organotin (the propyl derivatives of all methyl and n-butyl, mono- and di-n-octyl, and all phenyltin), range from 68–250 fg absolute and are in the pg l−1 range for 80 ml water samples extracted with 1 ml of solvent. The instrumental repeatablility is about 2% uncorrected and the linearity ranges to 5 ng absolute. This interface design is potentially useful for coupling more than one GC with one ICP-MS, for other plasma spectrometers (ICP-AES), for other types of large volume injection, and for high temperature (HT)-GC-ICP-MS.

Validated capillary gas chromatographic–mass spectrometric assay to determine 2-methylcitric acid I and II levels in human serum by using a pulsed splitless injection procedure

Background: Despite some clinical applications of 2-methylcitric acid (2-MCA) determination in urine and amniotic fluid, a diagnostic use of 2-MCA levels in serum is not common practice. This could be related to the complexity of the assay, or possibly to unawareness of other feasible clinical applications. Methods: The levels of the diastereomers 2-MCA I and II in human serum were determined by GC–MS based on a method using a pulsed splitless injection technique. A stable isotope dilution principle was modified considering the diastereomer ratio and impurities of the internal standard. Precision parameters as well as recovery rates of the assay were determined. Reference intervals for 2-MCA total, 2-MCA I and II levels were obtained in 52 healthy volunteers (31 female, 21 male, mean age 41.7±14.4 years). Results: 2-MCA was readily detected in each sample of serum, as well as in urine, cerebrospinal fluid and amniotic fluid. The limit of detection was 10 nmol/l for 2-MCA total. The internal standard showed a diastereomer ratio of 2-MCA II-d 3 to 2-MCA I-d 3 of 0.83±0.05, its chemical purity had to be corrected to 90.5±0.5%. In concentrations of 446, 750 and 1256 nmol/l 2-MCA total, recovery rates of 98.5, 93.7 and 88% with a mean intra-assay RSD of 1.5% were determined. The day-to-day precision was 10% RSD (SD 40 nmol/l) for 2-MCA total obtained with a pooled serum sample at a concentration of 401 nmol/l 2-MCA total over a period of 5 months ( n=17). The normal range for 2-MCA total in human serum was calculated as 81–266 nmol/l confirming previous findings. Conclusions: The GC–MS assay using a pulsed splitless injection procedure ensures a good response to differing concentrations of 2-MCA in various specimens. Considering exact determination of the diastereomer ratio as well as the purity of the internal standard, the assay offers good precision and recovery for 2-MCA I and II levels in serum.

Optimisation of a programmed split–splitless injector in the gas chromatographic–mass spectrometric determination of organochlorine pesticides

Large-volume injection techniques in gas chromatography are used to compensate for the at times limited detection sensitivity of mass spectrometric detection. In this work a programmed split–splitless injector in solvent split mode was employed to determine organochlorine pesticides in environmental samples. The injection conditions were selected by a Plackett-Burman design followed by a central composite design. The LODs obtained in the optimum conditions were compared with those obtained with splitless-MS and splitless-ECD. Finally, the method was applied to a soil sample.

Determination of environmental pollutants in soil and sediments - Some aspects of sample clean-up and GC analysis

The purpose of this work was to evaluate the results (especially calibration data) of gas chromatographic analysis of a wide range of pollutants (PAH, PCB, various groups of pesticides) in sediments in order determine the influence of any matrix present in the extract. Either crude extract or cleaned-up extract was used in the experiments. The so-called matrix-induced chromatographic response enhancement and its influence on the calibration of chromatographic systems were evaluated. Matrix present in an extract either increases or decreases the chromatographic response. It was shown that calibration procedures using a solution prepared in a matrix-free solvent often lead to systematic errors when only trace levels of analytes are present in the extract. The greater the extent of sample clean-up, the less frequent the unpredictable effects. After extensive clean-up the use of standards prepared in a “pure” solvent as the reference during calibration step can be accepted. Some conclusions for using different sleeves are given and critical points relating to the use of a splitless injector are mentioned.

Identification of bacteria from single colonies by fatty acid analysis

While a commercially available system for microbial identification by fatty acid analysis (Microbial Identification System, MIDI, Newark, DE, USA) is available, it requires approximately 40-mg wet weight of biomass. Various authors have published methods for fatty acid analysis of single colonies, but no database of organisms has been developed for those methods. A modification of the MIDI system to increase sensitivity while maintaining relative retention times and peak areas allows the standard peak naming tables and libraries of organisms to be used with single colonies. Samples are evaporated down before injection to concentrate the fatty acids, while splitless injection is used to allow more sample to enter the gas chromatograph. Several known bacteria were correctly identified by this method.

The Two Options for Sample Evaporation in Hot GC Injectors: Thermospray and Band Formation. Optimization of Conditions and Injector Design

Although classical split and splitless injection is more than 30 years old, we only start to understand the vaporization process in the injector. Solvent evaporation determines much of the process and is the first obstacle to overcome. Videos recorded on devices imitating injectors showed that sample (solvent) evaporation is often a violent process which is poorly controlled and might well explain many of the puzzling quantitative results often obtained. We do not adequately take into account that two vaporization techniques are in use. Partial solvent evaporation inside the syringe needle (optimized as "hot needle injection") produces thermospray: the sample liquid is nebulized upon leaving the needle. The resulting fog is rapidly slowed and moves with the gas. Solute evaporation largely occurs from microparticles suspended in the gas phase. Empty liners are most suitable. Fast autosamplers suppress vaporization in the needle, i.e., nebulization, and shoot a band of liquid into the chamber that must be stopped by a packing or obstacles suitable to hold the liquid in place during the 0.2-5 s required for solvent evaporation. Solute evaporation largely occurs from the surfaces onto which the sample is deposited. Insights into these mechanisms help optimize conditions in a more rational manner. Methods should specify whether they were optimized and validated for injection with thermospray or band formation. The insights should also enable a significant improvement of the injector design, particularly for splitless injection.

Performance of programmed temperature vaporizer, pulsed splitless and on-column injection techniques in analysis of pesticide residues in plant matrices

A programmed temperature vaporizer (PTV) injection technique has been recently implemented in our laboratory. In present paper its performance is compared with other GC injection techniques commonly used in trace analysis of organic contaminants. Twenty-six pesticides representing different chemical classes were selected for the study. This group comprised compounds typically subjected to discrimination in the injection port of the gas chromatograph, e.g., polar organophosphorus pesticides and thermolabile carbamates. In the first set of experiments standards in pure solvent were injected into GC systems employing different types of injection, i.e., (i) on-column, (ii) pulsed splitless, (iii) PTV solvent split, (iv) PTV splitless, and the responses of analytes were compared. Discrimination of troublesome compounds was significantly decreased with the application of PTV solvent split injection. In the second set of experiments repetitive injections of purified wheat samples were performed, with aims to evaluate the long-term stability of responses, as well as matrix effects in different stages of system contamination for each injection technique. The tolerance of the GC system to co-injected matrix components was increased in the order: on-column<pulsed splitless<PTV solvent split technique. As regards matrix effects, these were suppressed considerably with the PTV solvent split technique in comparison with pulsed splitless injection. With the latter technique after 66 injections of wheat samples relative responses (apparent recovery) reached as much as 450% for some compounds, while with the application of PTV matrix effects did not exceed 200% under the same conditions.

Simultaneous determination of melatonin and pyridoxine in tablets by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method for the qualitative and quantitative determination of melatonin plus pyridoxine commercial tablets is described. Melatonin and pyridoxine were simultaneously determined by GC-MS after extraction from ground tablets with methanol and derivatization with N-methyl-N-N-trimethlylsilyltrifluoroacetamide (MSTFA). The mass chromatograms were generated using 232 m/z ion for melatonin and 280 m/z ion for pyridoxine, respectively. Splitless injection offers good reproducibility with a standard deviation of 2%. The developed method was applied to analyze the melatonin and pyridoxine content from two different tablet formulations. Also, recovery, detection and quantification limits are reported.

Mass spectrometric detection in narrow-bore (0.10 mm I.D.) capillary chromatography: Fast, sensitive and selective analysis of polychlorinated biphenyls

The combination of narrow-bore capillary gas chromatography with bench-top quadrupole mass spectrometric detection was evaluated for the determination of polychlorinated biphenyls (PCBs). The qualitative and quantitative performances of the system are illustrated by several analyses (PCB standards and human milk extracts). Capillary columns with different internal diameters (0.10, 0.18 and 0.22 mm, respectively) were compared for their ability to separate PCB congeners and the analysis time. Short run times (less than 7 min) were sufficient for complete separation of PCB congeners on a 0.10-mm internal diameter (I.D.) capillary column without any loss of resolution when compared with a 0.22 mm I.D. column. Good qualitative and quantitative data acquisition was possible with quadrupole mass spectrometer for run times of 8 min, but incomplete peak reading was observed when run times were reduced to 3–4 min. Selected ion monitoring and dwell times of 10 ms are necessary to obtain detection limits for individual PCB congeners as low as 0.4 pg μl −1 for standard solutions and 0.2 ng g −1 fat for milk extracts. By using cold splitless injection, relatively high volumes (1 μl) for narrow-bore capillaries could be injected without any peak distortion.

Application of programmed-temperature split/splitless injection to the trace analysis of aliphatic hydrocarbons by gas chromatography

The dependence of the programmed-temperature solvent split sampling technique using a PSS (programmed-split/splitless) injection mode on different variables affecting the introduction of large sample volumes for a mixture of alkanes in capillary GC was evaluated. Apart from the studies found in the literature on different factors such as speed of injection, presence of adsorbent in the liner, internal diameter of the liner, initial and final injector temperature, split flow-rate and initial split time, affecting the chromatographic signal of different compounds, others were studied whose influence has not been considered until now. They include length of the microsyringe needle, adsorbent distribution in the liner, injection volume on analyte discrimination, speed of injector heating, time which the column stays at the initial temperature and time that the injector stays at the final temperature. Once finalised, the study of the PSS injection mode was compared with the conventional mode of gas chromatography splitless injection, and found that the proposed method increases sensitivity in GC trace analysis. Finally, the application of both injection modes in the determination of aliphatic hydrocarbons was tested in an atmospheric particulate sample.

Optimization and application of the PTV injector for the analysis of pesticide residues

The applicability of PTV splitless and solvent vent injection to the gas chromatographic analysis of 26 pesticides representing different chemical classes was evaluated. All parameters related to optimum injector performance in PTV splitless and PTV solvent vent (split vent) injection modes were tested. For PTV splitless injection of small sample volumes (1 μL), the inlet temperature program (initial inlet temperature, heating rate, final temperature), splitless time, and starting oven temperature were optimized. Parameters identified as optimal were then applied in PTV solvent vent injection. In the case of the PTV solvent vent technique, all injections were performed with pesticides dissolved in a binary mixture of cyclohexane-ethyl acetate (1 : 1, v/v). This solvent is used as a mobile phase in the HPGPC clean-up step involved in our multi-residue method. For the PTV solvent vent technique the following parameters were tested: maximum single injection volume, inlet temperature, vent flow, vent time, and vent period were determined for a single injection of 10 μL of sample. Thermodegradation and/or adsorption of some pesticides occurred as long as glass wool packed liner was used. To achieve lower detection limits, multiple injection concept was employed: 30 μL of standard solution were injected in three subsequent steps. Under optimized conditions even higher sample volumes could be injected. Good responses were obtained also for the compounds possessing relatively higher volatility compared to other tested analytes.

Quantitative Determination of Plasma C8–C26 Total Fatty Acids for the Biochemical Diagnosis of Nutritional and Metabolic Disorders

We have developed a capillary gas chromatography–electron-capture negative-ion mass spectrometry (GC/MS) method for the quantitative determination of C8–C26 total fatty acids in plasma. Following hydrolysis, hexane extraction, and derivatization with pentafluorobenzyl bromide, fatty acid esters are analyzed in two steps: a splitless injection and a second, split injection (1:100) for the quantitation of the more abundant long-chain species. Fourteen saturated and 25 unsaturated fatty acids are quantified by selected ion monitoring in ratio to 13 stable-isotope-labeled internal standards. Calibrations exhibit consistent linearity and reproducibility. Intraassay (n = 17) and interassay (n = 12) CVs ranged from 2.5 to 13.2% and from 4.6 to 22.9%, respectively. Recoveries ranged from 76 to 106%. Reference ranges were established for four age groups (<1 month, 1 month to 1 year, 1–17 years, >18 years) and compared to specimens from patients with nutritional deficiency of ω-3 and ω-6 polyunsaturated fatty acids, inborn errors of mitochondrial fatty acid oxidation, and peroxisomal disorders. Retrospective evaluation of the concentration of linoleic acid in 35 cases with a diagnosis of essential fatty acid deficiency previously made by gas chromatographic analysis with flame ionization detection (GC/FID) found a specificity and sensitivity of only 55 and 50%, respectively, for the GC/FID method when compared to GC/MS.

Speciation of butyltin compounds in marine sediments with headspace solid phase microextraction and gas chromatography mass spectrometry.

A method for the determination of organotin compounds (monobutyl = MBT, dibutyl = DBT, and tributyltin = TBT) in marine sediments by headspace Solid Phase Microextraction (SPME) has been developed. The analytical procedure involved 1) extraction of TBT, DBT and MBT from sediments with HCl and methanol mixture, 2) in situ derivatization with sodium tetraethylborate and 3) headspace SPME extraction using a fiber coated with poly(dimethylsiloxane). The derivatized organotin compounds were desorbed into the splitless injector and simultaneously analyzed by gas chromatography - mass spectrometry. The analytical method was optimized with respect to derivatization reaction and extraction conditions. The detection limits obtained for MBT, DBT and TBT ranged from 730 to 969 pg/g as Sn dry weight. Linear calibration curves were obtained for all analytes in the range of 30-1000 ng/L as Sn. Analysis of a standard reference sediment (CRM 462) demonstrates the suitability of this method for the determination of butyltin compounds in marine sediments. The application to the determination of TBT, DBT and MBT in a coastal marine sediment is shown.

Determination of Sucrose Esters of Fatty Acids in Food Additive Premixes by Gas Chromatography and Confirmation of Identity by Gas Chromatography/Mass Spectrometry

A gas chromatographic (GC) method was developed for the determination of sucrose monoesters of fatty acids (mono-SuE) and sucrose acetate isobutyrate (SAIB) in food additive premixes. Mono-SuE and SAIB fractions were prepared by column chromatography with either a C8 or a silica gel solid-phase extraction column. The mono-SuE fraction was acetylated and applied to a wide-bore GC column (0.53 mm x 15 m) by splitless injection for determination. The SAIB fraction was applied to the GC column without derivatization. Gas chromatography/mass spectrometry was used to confirm the identity of GC peaks. The detection limits for mono-SuE and SAIB were 0.005 and 0.01%, respectively. Mono-SuE (C12, C14, C16, C18, and C18:1) and SAIB were found in commercial food additive premixes and some foods.

Analytical Study of Comprehensive and Targeted Multidimensional Gas Chromatography Incorporating Modulated Cryogenic Trapping

This paper reports the results of an analytical study comparing capillary gas chromatography (GC) operated in the normal mode with 2 new GC techniques, comprehensive GC (GC x GC) and targeted (or selective) multidimensional GC, which use a longitudinally modulated cryogenic system (LMCS), recently developed in our laboratory. A high-temperature application of derivatized sterols, of interest in fecal pollution monitoring, was chosen for this work. A directly connected coupled-column ensemble was used, comprising a nonpolar column and a moderately polar column. With LMCS, effluent from the first column is zone-compressed in a cryogenic trap and then pulsed to a short second column, producing narrower peaks with sharp, tall peak responses at the detector. The modulator is operated at a constant frequency, e.g., 0.25 s(-1), to produce the GC x GC result, or is moved in a predefined manner so that whole peaks are selectively trapped and subsequently pulsed through to the second column in the targeted mode. Standard solutions containing a mixture of 7 sterols and 5-alpha-cholestane internal standard were used. Detection sensitivity is increased by a factor of >25 with the use of LMCS. The estimated limit of detection was about 0.1 microg/mL when normal GC with flame ionization detection (GC/FID) and a 1.0 microL splitless injection volume were used, compared with 0.02 and 0.004 microg/mL for the LMCS operated in GC x GC and selective modes, respectively. Calibration curves for GC/FID were linear over the 0.1-2.0 microg/mL range tested. Reproducibilities for the GC x GC and normal GC modes were comparable; generally, relative standard deviations (RSD) were on the order of 3-4%, based on raw peak responses. Improved reproducibility was found for selective LMCS operation, at an RSD of around 2%; with internal standardization, better results were achieved. The coupled-column arrangement allowed complete separation of sterol peaks from overlapping impurity peaks in a number of instances with LMCS modes, and its use should improve data quality over that of normal GC operation, in which the overlapping peaks interfere with measurement of peak response in the normal mode.

Monitoring of lipid degradation products by solid‐phase microextraction*

AbstractThe final sensory characteristic of vegetable oils is influenced by a series of aroma compounds and volatile oxidation products formed during storage and processing. Crude sunflower oil was stored at room temperature and was regularly refined by applying physical refining process in laboratory scale studying lipid degradation. Effect of long‐term storage and different steps of refining process on overall oxidative state and volatile compounds' formation was investigated by means of solid‐phase microextraction (SPME). Results were achieved by a 100‐μm polydimethylsiloxane fiber in the sample's headspace. Hexanal, tr‐2‐hexenal, tr‐2‐heptenal, tr‐2‐octenal, tr‐2‐nonenal, tr,tr‐2,4‐decadienal, α‐pinene, pentylfuran, 1‐octen‐3‐ol were used for external standard calibration. It was proved that hexanal and tr,tr‐2,4‐decadienal are aldehydes, which appear first in sunflower oil during long‐term storage. Detection limit using splitless injection and flame ionization detector was in 1–5 mg/kg depending on individual components. SPME was successfully applied for both controlling decomposition of hydroperoxides during bleaching and aldehydes removal during deodorization. SPME and traditional static headspace techniques were compared. Static headspace gave better recovery and lower detection limit for compounds having higher volatility, but SPME made it possible to measure higher molecular weight aldehydes providing a simple, reliable solution. © 2001 John Wiley &amp; Sons, Inc. J Micro Sep 13: 215–220, 2001

Improved Gas Chromatography–Mass Spectrometry Method for Simultaneous Identification and Quantification of Opiates in Urine as Propionyl and Oxime Derivatives

Several authors have reviewed existing methods (1)(2)(3)(4)(5)(6)(7)(8)(9) or presented new techniques (6)(7)(8)(9)(10)(11) for the analysis and separation of codeine, morphine, and the keto-opiates hydrocodone, hydromorphone, oxycodone, and oxymorphone. We present a modification of previously published procedures (6)(10) that incorporates the use of methoxyamine after enzymatic hydrolysis to form methoxime derivatives of the keto-opiates, which are then extracted using solid-phase columns and derivatized with propionic anhydride/pyridine. We used a gas chromatography–mass spectrometry system composed of a model 5890 gas chromatograph with splitless injection, a model 5970 mass-selective detector (both from Hewlett Packard), and a DB-5 capillary column [15 m × 0.25 mm (i.d.); 0.25 μm film thickness; JW helium (flow rate, 0.7 mL/min; linear velocity, 38 cm/s) was used as the carrier gas. The temperature program was as follows: initial temperature, 185 °C; ramp at 25 °C/min to 240 °C; hold for 0.5 min; ramp at 5 °C/min to 250 °C, then 40 °C/min to 290 °C; hold for 1.0 min. The injection temperature was 260 °C, and the transfer line temperature was 290 °C. The following were obtained from Radian Corporation: (a) codeine, morphine, hydrocodone, hydromorphone, and oxycodone, which were used to prepare calibrators; (b) deuterated codeine, morphine, hydrocodone, and hydromorphone, which were used as internal standards; and (c) oxymorphone and norcodeine, which were used for interference …

Possible interest of various sample transfer techniques for fast gas chromatography–mass spectrometric analysis of true onion volatiles

We improved GC–MS analysis of onion volatiles by comparing organic solvent partition with solid-phase microextraction (SPME) following cryo-trapping isolation and by comparing the same extraction methods on direct onion juice. Cryo-trapping produces very small quantities of volatiles and therefore is not a suitable extraction method for GC–MS analysis. We confirm that SPME accelerates the degradation of labile thiosulfinates but the lacrymatory factor remains intact. The identification of Allium thiosulfinates is only obtained on juice extracted by diethyl ether using a fast GC–MS analysis on a 10 m X 0.3 mm column of 4 μm coating, with routine splitless injection. The lacrymatory factor is best analysed directly on fresh onion juice by SPME with the same chromatographic conditions. To characterise and to quantify all the true onion volatiles, we propose to analyse the same sample by successive SPME–GC–MS and solvent extraction–GC–MS.