Optimal estimation of intensity of noisy peaks by matched filtering with application to chromatography : Part 2. Program description and simulation experiments

The (digital) matched filter described in Part 1 is applied to chromatographic data files. Practical aspects concerning the use of this filter are discussed. A FORTRAN program for interactive application of the matched filter is described. This program was used for simulation experiments in order to assess the model errors. Results indicate only a weak dependence on the signal-to-noise ratio. Selection of a 1/f noise model is recommended when the type of the signal noise is unknown. The relation between the Gamma and Guassian functions used is shown.     

Metrics of separation performance in chromatography. Part 1. Definitions and application to static analyses

Earlier introduced metrics of separation performance are described in a systematic way. After providing the definitions of the metrics suitable for a broad variety of applications, the study focuses on static analyses (isothermal GC, isocratic LC, etc.) and their general separation performance. Statistically expected number of resolved (adequately separated) single-component peaks is treated as the ultimate metric of general separation performance of chromatographic analysis. That number depends on the peak capacity of the analysis and the number of components in a test mixture. The peak capacity, in turn, depends on the separation capacity of a column and the lowest separation required by the data-analysis system for resolving poorly separated peaks. The separation capacity is a special case of a broader metric of the separation measure which is a function of column efficiency, solute separability, and the level of the solute interaction with a column stationary phase. The formulae for theoretical prediction of all these metrics for arbitrary pairs of peaks in static analyses are derived. To provide a better insight into the basic metrics of the separation performance, additional metrics such as the solute discrimination (relative difference in solute velocities), utilization of separability (solute discrimination per unit of their separability), specific separation (the separation per unit of separability), and others are defined and found for static analyses.     

HPLC of fatty acid esters of mono- and polyhydric alcohols. Part 1: Analytical separation

This paper shows how simply and yet very rapidly fatty acid esters of monohydric alcohols, but particularly partial and full fatty acid esters of fully hydric alcohols can be separated and determined by means of high-pressure liquid chromatography on LiChrosorb RP-8 with methanol/water. We have separated quantitatively the methylesters of the fatty acids C_8:0 to C_22:0 and C_24:0, the i-propyl-, i-butyl-, n-hexyl- and i-octyl-esters of the even-numbered fatty acids C_8:0 to C_18:0, mono- and difatty acid esters of the 1,3-bis-(2-hydroxyethyl)-5,5-dimethyl-hydantoin, mono-, di- and triesters of the trimethylolpropane as well as the tetraesters of the penta-erythrite.     

Separation of pesticides by capillary gas chromatography. Part 1: s-Triazines and substituted urea herbicides

The authors have shown that capillary columns are perfectly adequate for the separation of s-triazine herbicides and urea herbicides. The method presented remains adequate when the initial molecules are accompanied by some of their metabolites.     

Kinetic treatment of unsegmented flow systems : Part 1. Subjective and semiquantitative evaluations of flow-injection systems with gradient chamber

A variable-time kinetic model is used to evaluate a single-channel flow-injection system with gradient chamber that has been identified as a continuous-flow titration. A physical model, mathematical equations, computed concentration vs. time profiles, experimental data, and formal definitions are used to identify qualitative and quantitative features of the method that have not been apparent from the titration model for the system. It is shown that determinations can be performed with and without reactant in the flow stream and when reactant is in the flow stream, with and without reactant in the gradient chamber when the sample is introduced. It is shown that lowest concentrations with shortest cycle times can be achieved when determinations are performed without reagent in the gradient chamber initially. Characteristics unique to each of three different data processing options are used to evaluate the validity of equations presented. It is suggested that some methods previously identified as continuous-flow titrations are most accurately identified as variable-time kinetic methods, and it is shown that this semantic differentiation can provide improved insight into the methods and can expand the scope of the methods by suggesting new experimental approaches with potential advantages relative to previously described procedures.     

The theoretical basis of column chromatography in multicomponent separations. Part 1: Analytical requirements and peak resolution. Development of a high performance column system

The point of our published papers since 1957 is reviewed. The relations between the required value of peak resolution, K₁ (or R), and peak separation, K₃ (eqn 9); K₁ and relative accuracy of a peak height quantitative method, P_h (eqn. 10); K₁ and relative accuracy of a peak area method, P_a, (eqn. 12) at different concentration ratios, ?, are derived. The final result in Table 2 shows a large influence of ? on the required value of K₁. The approximately linear relation between peak width and retention value (eqn. 18) exists not only in GC. but also in HPLC. Plate height values H¹ and H^∞ for a solute with capacity ratio, k′, equal to unity or approaching infinity, respectively, are used to evaluate the column efficiency (eqn. 20). The measuring methods (eqn. 21,22,23) and parameters effecting on H¹ and H^∞ are given for GC packed column (eqn. 24), GC open tubular column (eqn. 25) and HPLC (eqn. 26). In the light of this, columns of high efficiency were developed. Some typical chromatograms for high speed analysis and separation of complex mixtures are given.     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies.

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane-isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a muPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3-300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been successfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at -20 degrees C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.     

Determination of bromazepam in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.     

Use of reference pools to compare the qualitative and quantitative determination of polychlorinated biphenyls by packed and capillary gas chromatography with electron capture detection. Part 1. Serum

Serum for reference pools of in vivo polychlorinated biphenyls (PCBs) was obtained from four goats that had received one dose (100 mg kg-1) of a selected technical Aroclor (AR) (1016, 1242, 1254 or 1260) and were allowed to recover for 30 d. These pools were used to assess the differences in an analytical method that determines and quantifies PCBs using packed-column gas chromatography (PCGC) (quantified on the basis of mean mass percent. data for grouped PCB peaks) and capillary-column gas chromatography (CCGC) (quantified on the basis of percent. composition data for specific congeners). With CCGC, results were statistically significantly different (p less than or equal to 0.0002) from results with PCGC for ARs 1016, 1242 and 1254 but not for AR 1260 (p = 0.23). When comparing these gas chromatographic methods using bovine serum spiked in vitro with the same ARs at 17-25 p.p.b., it was found that the methods were not statistically significantly different for any of the ARs (p = 0.30-0.92). Levels of serum PCB determined by the two methods for 12 persons, divided into two groups according to exposure, were compared using the paired t-test. Group 1 consisted of three persons with dietary and/or environmental exposure; one with dietary and/or environmental exposure in addition to occupational exposure dating back 20 years. Group 2 consisted of eight persons with recent occupational exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

High performance liquid chromatography with electrochemical detection. 1: Separation of Cannabis constituents and quantitation of Δ9 tetrahydrocannabinol

A simple, isocratic high performance liquid chromatography system (HPLC) with electrochemical detection (EC) was used to study Cannabis constituents. Several Cannabis extracts and reference standards were examined. A total of eleven constituents were separated, and three major cannabinoids; Δ⁸, Δ⁹ tetrahydrocannabinol (THC) and cannabidiol (CBD) were identified. A linear relationship was established for the quantitation of the halucinogenic constituent Δ⁹ THC. Minor contaminants in Δ⁹ THC reference standard, which were not detected by gas chromatography (GC) were detected for the first time. The detection limits of Δ⁹ THC and related cannabinoids were in the low nanogram range (2–20 ng).     

Determination of sparstolonin B by ultra‐high performance liquid chromatography coupled with triple quadrupole mass spectrometry: application to pharmacokinetic study of sparstolonin B in rat plasma

Sparstolonin B (SsnB), a spontaneous isocoumarin compound isolated from the tuber of Scirpus yagara Ohwi. (Cyperaceae), possesses potent anti‐inflammatory and antitumor activity. In the present study, a rapid and simple UHPLC/MS/MS method for determination of SsnB in rat plasma was developed and validated. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate containing rhein as an internal standard and separated on a C₁₈ column at 35 °C, with a gradient mobile phase consisting of acetonitrile and water containing 0.2% (v/v) formic acid within 2.1 min. MS/MS detection was accomplished in multiple reaction monitoring mode with negative electrospray ionization. The precursor–product ion transitions were m/z 266.9 [M–H]^? → m/z 211.0 for SsnB and m/z 283.2 [M–H]^? → m/z 239.0 for IS. The intra‐ and inter‐day precision (RSD) was <8.98% and the accuracy (RE) ranged from ?7.40 to 4.50%. The extraction recoveries ranged from 96.28 to 97.30%. The pharmacokinetic parameters were calculated using Win Nonlin53 software. The absolute bioavailability of SsnB was estimated to be 6.98%. The proposed method was successfully applied to a pharmacokinetic study of SsnB in rats after intravenous administration with a dose of 0.5 mg/kg and oral administration at a dose of 5 mg/kg. Copyright © 2015 John Wiley & Sons, Ltd.     

The improvement of SIMCA classification by using kernel density estimation : Part 1. A new probabilistic classification technique and how to evaluate such a technique

One of the disadvantages of SIMCA pattern recognition is its inability to produce probabilistic classifications. Attempts to correct this involve distributional assumptions. It appears that SIMCA can handle the residual error terms efficiently, but that inside the class model subspace a crude truncation is used for determining a “normal range”, inside which all points are treated as equal. An improvement is made by applying kernel density estimation to the scores inside the class model subspace in combination with a normal error distribution in the remaining dimensions (CLASSY method). The evaluation of these probabilistic classification methods is discussed theoretically.     

Comparison of flux approximations in electrochemical digital simulation. Part 2: Complications due to homogeneous chemical reactions, charge estimation and application to the ultramicrodisk electrode

In numerical simulation of electrochemical systems the estimation of current flow or passed charge from simulated concentrations plays a crucial role and various approaches within the framework of finite differences are examined in this paper. Both on an exponentially expanding grid in X-space, and the corresponding equidistant grid in transformed Y-space, fluxes were expressed as either gradient approximations using a variable number of points, or as the spatial integral of rates of changes of the concentrations with time over the whole diffusion space, in the presence of homogeneous chemical reactions. Also, the passed charge was computed for these cases. The cases of chronoamperometric boundary conditions at a shrouded plane and an ultramicrodisk electrode (UMDE) were simulated. Results show that in all cases, fluxes computed on the basis of the spatial concentration gradient at the electrode are the most accurate provided that the Rudolph modification of the two-point flux in Y-space is used; it also works very well in the presence of homogeneous chemical reactions. The flux approximation on the basis of spatial integration of the time derivative of concentration comes a close second in Y-space in the case of one-dimensional simulations when K is not too large, and also works rather well in the case of the UMDE in transformed space. Charge computed as the time integral of flux can be quite accurate if two separate simulation runs are carried out, combining the results by extrapolation. Charge computed as the spatial integration of concentration loss in the diffusion space (not practical in the presence of a homogeneous chemical reaction) can also be quite accurate, both in the one- and two-dimensional case.     

Determination of cefuroxime lysine in rat brain microdialysates by ultra‐fast liquid chromatography with UV and tandem mass spectrometry: application to an acute toxicokinetic study

Cefuroxime lysine is a new second‐generation cephalosporins, which can penetrate the blood–brain barrier to cure the meningitis. In order to investigate its acute toxicokinetic study after intraperitoneal injection of 675 mg/kg cefuroxime lysine, a sensitive and clean ultra‐fast liquid chromatography–tandem mass spectrometry (UFLC‐MS/MS) method for the determination of cefuroxime lysine in microdialysate samples was developed and validated, which was compared with UFLC‐UV as a reference method. Chromatographic separation was performed on a Shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 µm), with an isocratic elution of 0.1% formic acid in acetonitrile–0.1% formic acid in water (45:55, v/v) for LC‐MS and acetonitrile–20 mm potassium dihydrogen phosphate (pH 3.0,20:80, v/v) for LC‐UV. The lower limit of detection was 0.01 µg/mL for LC‐MS and 0.1 µg/mL for LC‐UV method, with the same corresponding linearity range of 0.1–50 µg/mL. The intra‐ and inter‐day precisions (relative standard deviation) for both methods were from 1.1 to 8.9%, while the accuracy was all within ±10.9%. The results of both methods were finally compared using paired t‐test; the results indicated that the concentrations measured by the two methods correlated significantly (p < 0.05), which suggested that the two methods based on LC‐MS and LC‐UV were suitable for the acute toxicokinetic study. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of didanosine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A method based on solid-phase extraction coupled to liquid chromatography with positive ion electrospray ionization and tandem mass spectrometric detection was developed for the determination of didanosine in human serum, using lamivudine as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions m/z 237 --> 136.7 for didanosine and m/z 230 --> 111.7 for lamivudine. The method was linear over the range studied (10-1500 ng ml(-1)), with r(2) > 0.98, and the run time was 5 min. The intra- and inter-assay precisions were < or =10% and the intra- and inter-assay accuracies were >95%. The absolute recoveries were 99.8% (10 ng ml(-1)), 98.4% (30 ng ml(-1)), 91.5% (700 ng ml(-1)) and 94.7% (1200 ng ml(-1)). The limits of detection and quantitation were 5 and 10 ng ml(-1), respectively. The method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (200 mg) of reference and test didanosine formulations (buffered powder for oral solutions), in an open, two-way, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (area under the serum concentration versus time curve from time zero to infinity) were within the range 80-125%, which supports the conclusion that the two formulations are bioequivalent regarding the rate and extent of didanosine absorption.     

Characterization of new types of stationary phases for fast and ultra-fast liquid chromatography by signal processing based on AutoCovariance Function: A case study of application to Passiflora incarnata L. extract separations

In this study, a comparative investigation was performed of HPLC Ascentis^® (2.7 μm particles) columns based on fused-core particle technology and Acquity^® (1.7 μm particles) columns requiring UPLC instruments, in comparison with Chromolith™ RP-18e columns. The study was carried out on mother and vegetal tinctures of Passiflora incarnata L. on one single or two coupled columns. The fundamental attributions of the chromatographic profiles are evaluated using a chemometric procedure, based on the AutoCovariance Function (ACVF). Different chromatographic systems are compared in terms of their separation parameters, i.e., number of total chemical components (m_tot), separation efficiency (σ), peak capacity (n_c), overlap degree of peaks and peak purity. The obtained results show the improvements achieved by HPLC columns with narrow size particles in terms of total analysis time and chromatographic efficiency: comparable performance are achieved by Ascentis^® (2.7 μm particle) column and Acquity^® (1.7 μm particle) column requiring UPLC instruments. The ACVF plot is proposed as a simplified tool describing the chromatographic fingerprint to be used for evaluating and comparing chemical composition of plant extracts by using the parameters D% – relative abundance of the deterministic component – and c_EACF – similarity index computed on ACVF.     

Analysis of polybrominated diphenyl ethers (PBDEs) by liquid chromatography with negative-ion atmospheric pressure photoionization tandem mass spectrometry (LC/NI-APPI/MS/MS): application to house dust

Eight polybrominated diphenyl ether (PBDE) congeners of primary interest to the US EPA were separated using reverse-phase liquid chromatography on an octadecylsilane column. BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183, and BDE-209 were baseline-resolved under isocratic conditions in 92:8 methanol/water (v/v). Negative-ion atmospheric pressure photoionization (NI-APPI) with a toluene dopant produced precursor ions corresponding to [M–Br+O]^– for the eight congeners studied. Each congener was quantified by tandem mass spectrometry through a unique multiple reaction monitoring (MRM) transition. On-column limits of detection were between 2.4 and 27.8 pg for the eight congeners studied, with an intra-day method precision of 9%. The LC/NI-APPI/MS/MS method was validated for the analysis of the eight PBDE congeners in NIST SRM 2585 (Organics in House Dust). Pressurized liquid extraction (PLE) with subsequent LC/NI-APPI/MS/MS analysis afforded quantitative recovery for all eight PBDE congeners with recoveries ranging from 92.7 to 113%. The liquid-phase separation of the LC/NI-APPI/MS/MS method is not prone to the thermal degradation issues that plague splitless GC based analyses of highly brominated PBDEs such as BDE-209.     

Simultaneous separation of angiotensin and endothelin peptide families by high-performance liquid chromatography: application to the specific radioimmunoassay measurement of angiotensin II or endothelin-1 from tissue

Currently available measurements of endogenous angiotensin II (ANG II) and endothelin-1 (ET-1) concentrations by radioimmunoassay (RIA) lack specificity to ANG II or ET-1. ANG II and ET-1 antibodies cross-react with immuno-reactive angiotensin and endothelin family members, respectively. We have therefore developed an ion-pair reversed-phase high-performance liquid chromatography (HPLC) for simultaneously separating angiotensin and endothelin peptides and enhancing RIA specificity in the measurement of ANG II and ET-1. The developed HPLC separation was applied to canine myocardium extracts; ANG II or ET-1 fractions were collected and quantified by RIA. Elution times for both peptide families, ANG I, ANG II, ANG III, ANG IV, ANG II(4–8), bET-1, ET-1, ET-2 and ET-3 were within 25 min. In normal canine myocardium from the right atrium, right ventricle, left atrium and left ventricle, ANG II concentrations were 39±11, 28±21, 31±11 and 21±8 fmol/g and ET-1 concentrations were 43±16, 42±19, 55±21 and 57±34 fmol/g (mean±SD, N=7), respectively. The combination of HPLC with RIA renders the measurement of ANG II or ET-1 specific and convenient, and saves time. This HPLC separation may be applied to the specific measurement of other immuno-reactive angiotensin and endothelin peptides.     

Development of a 6-hydroxychroman-based derivatization reagent: application to the analysis of 5-hydroxytryptamine and catecholamines by using high-performance liquid chromatography with electrochemical detection

We developed a novel derivatization reagent, (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (alpha-CA), which exhibits intense EC response. NPCA successfully yielded alpha-CA derivatives of primary amines by a two-step derivatization procedure. Following pre-column derivatization with NPCA, a simultaneous determination of alpha-CA derivatives of neuroactive monoamines [dopamine (DA), epinephrine, and 5-hydroxytryptamine (5-HT)], their monoamine oxidase metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid) and their catechol-O-methyltransferase metabolites [3-methoxytyramine (3-MT) and normetanephrine (NMN)] was completely achieved using our HPLC-EC method. Using an HPLC equipped with coulometric electrode-array detection system, the resultant alpha-CA derivatives of NMN, 5-HT, DA and 3-MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4-fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16-60 fmol on column (signal-to-noise ratio 3). The proposed HPLC method was applied to determine 5-HIAA, HVA, alpha-CA-5-HT and alpha-CA-DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions.