Prediction of precision from signal and noise measurement in liquid chromatography: Limit of detection

Summary A method to determine the limit of detection (LOD) in high performance liquid chromatography (HPLC) is described. The power spectral density of instrumental baseline variation is fitted by the simplex least squares methods with a mixed random process of white noise and Markov process as a model. The white noise is characterized by standard deviation (SD), w; the Markov process by the SD, m, and auto-correlation degree, . All required parameters for calculating the LOD signal are obtained by experiment without repeat measurements. No arbitrary constants are needed. The LOD signal is uniquely determined and is characterized by 33.3% relative standard deviation (RSD) of analyte measurements and 0.13% of the error of the first type. This signal also specifies that the signal-to-noise ratio=3, using the definition of noise originating from the white noise and Markov process. The theoretical conclusion is verified by the Monte Carlo simulation using real baseline and peaks. The LOD concentrations for naphthalene, acenaphthene, pyrene and perylene are given.First part of series cited as Ref. [1].     

Prediction of precision from signal and noise measurement in liquid chromatography: Mathematical relationship between integration domain and precision

Summary The precision of integration over noisy instrumental output for quantitative analysis is studied. A probability theory is developed to predict the relative standard deviation (RSD) of integration results over an integration domain from one-point integration (peak height measurement) to entire area integration in HPLC. Common integration modes of horizontal zero line and oblique zero line are taken into account, but no peak overlap is assumed. The question of the analytical superiority of peak height measurement or integration for quantitation is answered. In the HPLC apparatus used, the minimum RSD of measurements is found in the integration domain of ca. ±0.5 σ for analytes [peaks are approximated by the Gaussian signal of width, σ (standard deviation)]. The RSD of integration measurements is also shown to depend on the stochastic properties of background noise (uncorrelated noise and correlated 1/f type noise). The theoretical conclusion is verified by Monte Carlo simulation and HPLC experiments for some aromatic compounds. Second Part of series cited as Ref. [1].     

Prediction of precision from signal and noise measurement in liquid chromatography: Mathematical relationship between integration domain and precision

Summary The precision of integration over noisy instrumental output for quantitative analysis is studied. A probability theory is developed to predict the relative standard deviation (RSD) of integration results over an integration domain from one-point integation (peak height measurement) to entire area integration in HPLC. Common integration modes of horizontal zero line and oblique zero line are taken into account, but no peak overlap is assumed. The question of the analytical superiority of peak height measurement or integration for quantitation is answered. In the HPLC apparatus used, the minimum RSD of measurements is found in the integration domain of ca. ±0.5 for analytes [peaks are approximated by the Gaussian signal of width, (standard deviation)]. The RSD of integration measurements is also shown to depend on the stochastic properties of back-ground noise (uncorrelated noise and correlated 1/f type noise). The theoretical conclusion is verified by Monte Carlo simulation and HPLC experiments for some aromatic compounds.Second Part of series cited as Ref. [1].     

Evaluation of the signal intensity of induced peaks in indirect photometric detection of nonelectrolytes in reversed-phase high-performance liquid chromatography

Summary We have experimentally evaluated the equation representing the peak area of induced peaks in indirect photometric detection of nonelectrolytes as a function of the amount and concentration of the analyte and the visualizing agent and their capacity factor, the phase ratio of the column and the absorptivity and the pathlength of detection. Aliphatic alcohols and hydrocarbons were used as the test analytes. Most of the analytes behaved according to the relationship expressed by the equation.     

Refractive index detection in microbore column liquid chromatography

Summary An existing commercial refractive index detector was modified for use with microbore column LC systems. The detector utilizes the Fresnel method. The effect of band dispersion and dilution at the detector side is of extreme importance in connection with the miniaturized LC system. In the modified model the original heat-exchanger tube was removed and a stainless steel capillary was used for heat-exchanging. Gaskets having different cell volumes were also examined with respect to band broadening and sensitivity. The detection limit was 10ng for di-n-pentyl phthalate. The examples include the detection of phthalates, alcohols, n-paraffins, and kerosine.     

Sensitive determination of cinnarizine in human plasma by high performance liquid chromatography and fluorescence detection

Summary A sensitive high performance liquid chromatographic method has been developed for the determination of cinnarizine in human plasma. Cinnarizine and clocinizine (internal standard) were extracted from acidified plasma (pH 4.7) into carbon tetrachloride and the organic layer was evaporated. The products were separated on a Microspher C18 (3 m) column, using a mixture of 0.04 % triethylamine in 0.01 M ammonium dihydrogen phosphate (NH₄H₂PO₄), pH adjusted to 4.2 with orthophosphoric acid (H₃PO₄), and acetonitrile (2080, v/v) as mobile phase, at a flow rate of 1 ml/min at 40°C. Fluorescence detection (_ex = 245 nm, _em = 310 nm) was used; the detection limit was 0.5 ng/ml under the conditions used, and the calibration curve linear in the concentration range evaluated (1–60 ng/ml). The assay has been used to measure cinnarizine concentrations in plasma after oral administration to volunteers.     

Simulation of induced peaks in indirect detection of non-electrolytes in high-performance liquid chromatography

Summary Induced peaks observed in indirect detection of nonelectrolytes in high-performance liquid chromatography are simulated. The equation representing the signal intensity of the induced peaks is derived, and it is verified by the experimental results. The peak area is proportional to (K _b +1) k _a /| _a —k _b |, where k _a ad k _b are the capacity factors of the visualization agent and the analyte, respectively.     

Prediction of the detection ranges for HPLC-peroxyoxalate chemiluminescence detection system of fluorescent compounds

Summary A manual method for predicting the detection ranges of fluorescent compounds for the HPLC-peroxyoxalate chemiluminescence detection system is presented utilizing bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) as a chemilumigenic reagent. The generated chemiluminescence decay curves were measured on a photomultiplier and extrapolated to zero time based on the first part of the decay curve. Dipyridamole, perylene, DNS-Ser, Rose Bengal, DNS-Asp, Trp-P-1, pyrencarboxylic acid methyl ester, perfenazine, alimemazine, oxypertine, Glu-P-2, benzydamine and doxorubicin gave chemiluminescence intensity (Icl) values of 3.2 × 10⁶, 2.8 × 10⁶, 2.3 × 10⁵, 2.1 × 10⁵, 1.9 × 10⁵, 1.4 × 10⁵, 9.4 × 10³, 6.2 × 10³, 4.2 × 10³, 3.3 × 10³, 2.3 × 10³, 9.7 × 10² and 3.1 × 10², respectively. The reaction conditions for the HPLC-CL detection system were investigated and optimum conditions obtained.     

Determination of urinary 5-S-cysteinyldopamine by high-performance liquid chromatography with electrochemical detection

Summary 5-S-Cysteinyldopamine, a new metabolite of dopamine, was determined in urine by high-performance liquid chromatography with electrochemical detection. The catechol was detected in 14 of 21 melanoma patients and 7 of 21 normal subjects; the highest values were 657 μg/day for melanoma patients and 44 μg/day for normal subjects. These results suggest that the cysteine conjugate may arise from autoxidation of dopamine but tyrosinase may also participate in the oxidation.     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for cations on polystyrene polymers

Summary The distribution equilibria of cationic compounds in reversed-phase chromatographic systems (ion-pair chromatography) have been studied on the basis of their effect on a detectable mobile phase component. The solid phase was a polystyrene-divinylbenzene copolymer and the detectable component, a quaternary ammonium ion, 1-methylpyridine. The solutes were mono- and divalent amines and quaternary ammonium ions. The cations can be retained by ion-pair adsorption and ion exchange. Expressions for the ion-pair retention of the solutes and the mobile phase cation (system peak) have been developed assuming Langmuir distribution of ion pairs to a solid phase with one kind of binding site. The validity of the expressions has been tested by evaluation of ion-pair distribution constants using non-linear curve fitting techniques. Good agreement for the constants of common ion pairs was obtained from different kinds of capacity ratio expressions. Ion exchange retention can appear beside ion-pair retention, and it has been observed in the pH range 1.6–6.1. The effect depends not only on cations in the mobile phase, but also on the nature of the buffering systems.     

Determination of vitamin A in pharmaceutical preparations by high-performance liquid chromatography with diode-array detection

Summary A very simple high-performance liquid chromatographic method for determination of Vitamin A in pharmaceutical preparations without the need for saponification was developed. A reversed-phase (Nova-Pack C₁₈, 4 m) column was used with a mobile phase of acetonitrile-tetrahydrofuran-water (55378) and a flowrate of 1.5 ml/min. Sample treatment only consisted of the extraction of retinol acetate content from capsules or tablets with methanol. Total extraction was achieved by shaking vigorously with the aid of magnetic stirring for three hours at room temperature. No change of solvent is necessary to introduce the sample in the chromatographic system. This method is suitable for routine quantification of Vitamin A.     

Fluorometric detection with a packed flow cell in micro high-performance liquid chromatography

Summary A packed flow cell was used for fluorometric detection in micro high-performance liquid chromatography. The flow cell consisted of fused-silica tubing packed with the same material as the separation column. A focusing effect of the stationary phase on the signal intensity was observed, leading to an improvement of the mass detection limit, as achieved by on-column detection.     

Analysis of commercial ceramides by non-aqueous reversed-phase liquid chromatography with evaporative light-scattering detection

Summary This paper describes the development of a chromatographic system for analysis of commercial ceramides structurally similar to those found in the stratum corneum. The ceramides used in this study contain different amine based (phytosphingosine, sphingosine and dihydrosphingosine) and fatty acids of different chain lengths and with different functional groups (hydroxylated and unsaturated). Non-aqueous reversed-phase (NARP) liquid chromatography with evaporative light-scattering detection (ELSD) were the techniques chosen in accordance with the nature of the ceramides. The eluent strength and the potential selectivity of different organic solvents were investigated. On a C₁₈-bonded silica, the most promising chromatographic conditions employed a gradient from ACN-THF, 95∶5, to ACN-THF-PrOH, 35∶5∶60, in 15 min with a constant concentration of TEA (10 mM) and a stoichiometric amount of formic acid.     

Determination of clozapine in human plasma by solid-phase extraction and liquid chromatography with amperometric detection

Summary A sensitive and selective high-performance liquid chromatographic method has been developed for monitoring clozapine levels in human plasma. Chromatography was performed on a reversed-phase column (C₈, 150 mm×4.6 mm i.d., 5 μm) with acetonitrile-aqueous sodium acetate solution, 88∶12 (v/v), as mobile phase; the flow rate was 1 mL min⁻¹. Clozapine oxidation at +800 mV was detected amperometrically. Response was linearly dependent on concentration over the range 50–1500 ng mL⁻¹ clozapine in plasma. Sample preparation by solid-phase extraction before HPLC analysis gave high extraction yield (94%). The accuracy and precision of the method were both very good (recovery: 97%;RSD<3.3%).     

Structure of hydroxycinnamic acid derivatives established by high-performance liquid chromatography with photodiode-array detection

Summary The present paper describes the development of a method for the differentiation of hydroxycinnamic acid derivatives by HPLC with photodiode-array detection. Furthermore spectral data of the compounds under investigation are given. Whereas p-coumaric acid derivatives are distinguishable from caffeic and ferulic acid derivatives by the positions of their spectrum maxima and their convexity interval value, it is not possible to distinguish between caffeic and ferulic acid derivatives because of overlapping spectrum maxima and convexity interval values.     

Structure of hydroxycinnamic acid derivatives established by high-perfomance liquid chromatography with photodiode-array detection

Summary The present paper describes the development of a method for the differentiation of hydroxycinnamic acid derivatives by HPLC with photodiode-array detection. Furthermore spectral data of the compounds under investigation are given. Whereas p-coumaric acid derivatives are distinguishable from caffeic and ferulic acid derivatives by the positions of their spectrum maxima and their convexity interval value, it is not possible to distinguish between caffeic and ferulic acid derivatives because of overlapping spectrum maxima and convexity interval values.     

Direct analysis of industrial oligoglycerols by liquid chromatography with evaporative light-scattering detection and mass spectrometry

Summary A liquid chromatographic method has been developed for analysis of industrial polyglycerols, precursors of polyglycerol fatty esters, which are non-ionic surfacetants. The method utilizes two complementary chromatographic systems: porous graphitic carbon and an aminopropyl polymer with an acetonitrile-water mixture as mobile phase. Detection of these non-UV absorbing compounds was effected by means of evaporative light-scattering detection. Their structures were determined by comparison of their retention with that of synthesized standards, and by mass spectrometry.     

Determination of ambroxol in dog plasma by high-performance liquid chromatography and UV detection

Summary A new sensitive HPLC-UV method has been developed and validated for the determination of amboroxol in dog plasma enabling the investigation of a newly developed 75 mg ambroxol-containing retard capsule of EGIS Pharmaceuticals Ltd., Budapest, Hungary. A gradient method was used for removing the longer retained plasma components of no interest. The separation was performed on a BDS Hypersil C18 (5 μm, 250×2.1 mm) analytical column, supplied with a 10 mm guard column containing the same packing material. The detection was performed at 210 nm. The calibration curve was linear in the range 25–2000 ng·mL⁻¹. Nerisopam (EGIS-6775) was used as internal standard. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

The analysis of ethinyloestradiol in tablets by liquid chromatography with electrochemical detection

Summary The analysis of ethinyloestradiol in tablets has been carried out by reversed phase high performance liquid chromatography (HPLC) using electrochemical detection. The drug was eluted with aqueous acetonitrile containing sodium acetate as background electrolyte. The mobile phase was used as extracting solvent, and 4-tertpentylphenol was used as internal standard.     

Analysis of ofloxacin in plasma samples by high-performance liquid chromatography

Summary A sensitive HPLC method has been developed for determination of ofloxacin (OFL) in biological fluids. Sample preparation was performed by adding phosphate buffer (pH 7.4, 0.1m) then extraction with trichloromethane. OFL and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column with aqueous phosphate solution-acetonitrile, 80∶20, as mobile phase. The fluorescence of the column effluent was monitored at λ_ex 338 and λ_em 425 nm. The retention times were 2.66 and 4.24 min for OFL and SAR, respectively, and the detection and quantitation limits were 8 and 15 ng mL⁻¹, respectively. Plots of response against ofloxacin concentration were linear in the range 8 to 2000 ng mL⁻¹. Recovery was 92.9% for OFL.