A functional peptide-mediated colorimetric assay for mercury ion based on dual-modified gold nanoparticles.

In the work, a rapid and accurate biosensor for mercury ions (Hg2+) was constructed, with which aggregation of dual-modified (DGPFHR- and CALNN-) gold nanoparticles (D/C-AuNPs) could be triggered by the high specificity of peptides to Hg2+. The given peptide DGPFHR possesses great capability of capturing Hg2+, accompanied by the conformational folding. Under the circumstances, D/C-AuNPs were employed as the detection probes to accomplish the quantitative analysis of Hg2+. This is primarily because the specific Hg2+-induced folding of peptides reduces the electrostatic repulsion and steric hindrance, thus accelerating the AuNPs aggregation. The principle and application potential of this proposal was proved by evidence. And the results demonstrated that Hg2+ ions could be selectively detected as low as 28 nM with a linear range of 100-800 nM. In consideration of superior simplicity, selectivity, accuracy and stability, the protocol was advantageous over other projects in practical measurement of various water samples.

Ultrasensitive visual detection of Hg2+ ions via the Tyndall effect of gold nanoparticles.

This work reports a new methodology for naked-eye nanosensing of Hg2+ where the Tyndall effect of gold nanoparticles (GNPs) acts as a light scattering signalling readout. Its utility is demonstrated with ultrasensitive detection of the target with a limit down to 0.13 nM (∼5461-fold sensitivity improvement over conventional GNP-based methods with surface plasmon resonance signalling).

Vacuum Ultraviolet Absorption Spectroscopy Analysis of Breath Acetone Using a Hollow Optical Fiber Gas Cell.

Human breath is a biomarker of body fat metabolism and can be used to diagnose various diseases, such as diabetes. As such, in this paper, a vacuum ultraviolet (VUV) spectroscopy system is proposed to measure the acetone in exhaled human breath. A strong absorption acetone peak at 195 nm is detected using a simple system consisting of a deuterium lamp source, a hollow-core fiber gas cell, and a fiber-coupled compact spectrometer corresponding to the VUV region. The hollow-core fiber functions both as a long-path and an extremely small-volume gas cell; it enables us to sensitively measure the trace components of exhaled breath. For breath analysis, we apply multiple regression analysis using the absorption spectra of oxygen, water, and acetone standard gas as explanatory variables to quantitate the concentration of acetone in breath. Based on human breath, we apply the standard addition method to obtain the measurement accuracy. The results suggest that the standard deviation is 0.074 ppm for healthy human breath with an acetone concentration of around 0.8 ppm and a precision of 0.026 ppm. We also monitor body fat burn based on breath acetone and confirm that breath acetone increases after exercise because it is a volatile byproduct of lipolysis.

Direct optical detection of cell density and viability of mammalian cells by means of UV/VIS spectroscopy.

The critical process parameters cell density and viability during mammalian cell cultivation are assessed by UV/VIS spectroscopy in combination with multivariate data analytical methods. This direct optical detection technique uses a commercial optical probe to acquire spectra in a label-free way without signal enhancement. For the cultivation, an inverse cultivation protocol is applied, which simulates the exponential growth phase by exponentially replacing cells and metabolites of a growing Chinese hamster ovary cell batch with fresh medium. For the simulation of the death phase, a batch of growing cells is progressively replaced by a batch with completely starved cells. Thus, the most important parts of an industrial batch cultivation are easily imitated. The cell viability was determined by the well-established method partial least squares regression (PLS). To further improve process knowledge, the viability has been determined from the spectra based on a multivariate curve resolution (MCR) model. With this approach, the progress of the cultivations can be continuously monitored solely based on an UV/VIS sensor. Thus, the monitoring of critical process parameters is possible inline within a mammalian cell cultivation process, especially the viable cell density. In addition, the beginning of cell death can be detected by this method which allows us to determine the cell viability with acceptable error. The combination of inline UV/VIS spectroscopy with multivariate curve resolution generates additional process knowledge complementary to PLS and is considered a suitable process analytical tool for monitoring industrial cultivation processes.

Zinc(II) salphen complex-based fluorescence optical sensor for biogenic amine detection.

Biogenic amines have attracted interest among researchers because of their importance as biomarkers in determining the quality of food freshness in the food industry. A rapid and simple technique that is able to detect biogenic amines is needed. In this work, a new optical sensing material for one of the biogenic amines, histamine, based on a new zinc(II) salphen complex was developed. The binding of zinc(II) complexes without an electron-withdrawing group (complex 1) and with electron-withdrawing groups (F, complex 2; Cl, complex 3) to histamine resulted in enhancement of fluorescence. All complexes exhibited high affinity for histamine [binding constant of (7.14 ± 0.80) × 104, (3.33 ± 0.03) × 105, and (2.35 ± 0.14) × 105 M-1, respectively]. Complex 2 was chosen as the sensing material for further development of an optical sensor for biogenic amines in the following step since it displayed enhanced optical properties in comparison with complexes 1 and 3. The optical sensor for biogenic amines used silica microparticles as the immobilisation support and histamine as the analyte. The optical sensor had a limit of detection for histamine of 4.4 × 10-12 M, with a linear working range between 1.0 × 10-11 and 1.0 × 10-6 M (R2 = 0.9844). The sensor showed good reproducibility, with a low relative standard deviation (5.5 %). In addition, the sensor exhibited good selectivity towards histamine and cadaverine over other amines, such as 1,2-phenylenediamine, triethylamine, and trimethylamine. Recovery and real sample studies suggested that complex 2 could be a promising biogenic amine optical sensing material that can be applied in the food industry, especially in controlling the safety of food for it to remain fresh and healthy for consumption.

Online Monitoring of Solutions Within Microfluidic Chips: Simultaneous Raman and UV-Vis Absorption Spectroscopies.

Microfluidics is an appealing analytical tool in the global effort to close the nuclear fuel cycle. Using a microfluidic chip permits the analysis of greatly reduced sample volumes compared to what is necessary for traditional analytical methods. There is a commensurate reduction in disposal volume and cost. The development of novel sensors is necessary to take full advantage of the microchip configuration, where optical-spectroscopy-based approaches offer a powerful route to characterize chemical composition. This study uses simultaneously applied UV-vis and micro-Raman spectroscopies adapted to function on the microscale to analyze in situ both the Nd3+ (UV-vis-active) and HNO3 (Raman-active) concentrations in the same sample. An adjustable translation platform was designed to hold the micro-Raman probe above and perpendicular to the chip face and the UV-vis probe in the plane of the chip. These complimentary spectral techniques when processed through multivariate partial least-squares (PLS) models gave an accurate picture of the widely varying solution concentrations as a function of time for each solution component. Solution matrix effects can drastically alter analyte signatures as measured by both UV-vis absorbance and Raman spectroscopy. PLS methods successfully modeled these spectral changes and accurately measured concentrations of components of interest within the microfluidic chip.

UV-vis sensor array combining with chemometric methods for quantitative analysis of binary dipeptide mixture (Gly-Gly/Ala-Gln).

Many endogenous peptides are circulating in bodily fluids at micromole level, and accurate analysis of endogenous peptides at such low level is important. In this study, we presented an extensible, facile and sensitive sensor array based on UV-Vis spectroscopy of the AuNPs combined with chemometric methods for quantitative analysis of binary peptide mixture (Gly-Gly/Ala-Gln) using UV-Vis spectroscopy. High concentration arginine (Arg) and Cr3+ can induce aggregation of the AuNPs and DNA-AuNPs. However, the glycylglycine (Gly-Gly) and alanyl-glutamine (Ala-Gln) can prevent the AuNPs from aggregation. We investigated the prevention of AuNPs aggregation by using Gly-Gly and Ala-Gln mixtures and constructed sensor arrays for quantitative analyses of Gly-Gly and Ala-Gln mixtures. The color change of the solution is relevant to the dose of the target, and it can be visualized by the naked eyes or monitored by UV-Vis spectrometry. Results showed that the concentrations of Arg and Cr3+ are the key factors affecting the sensitivity of the sensor array. Whereas when Gly-Gly and Ala-Gln have to be analyzed simultaneously, concentrations of Arg and Cr3+ both for Gly-Gly and Ala-Gln are difficult to be optimized. Taking the advantages of multivariate analysis and data fusion, PLS models and backward interval PLS (BiPLS) models were built for fused dataset constructed by UV-Vis data obtained at different concentrations of Arg and Cr3+. The best results were obtained from the PLS models. The proposed method can be extended to analysis of other peptides in more complex mixture systems.

Development of an optical biosensor for the detection of Trypanosoma evansi and Plasmodium berghei.

A laboratory prototype system that correlates murine blood absorbance with degree of infection for Plasmodium berghei and Trypanosoma avensi has been designed, constructed and tested. A population (n = 6) of control uninfected, Plasmodium infected and Trypanosoma infected BALB/c mice were developed and spectral absorption measurements pre and post infection were made every 3 days. A fibre optic spectrometer set-up was used as the basis of a laboratory prototype biosensor that uses the Beer Lambert Law to relate Ultraviolet-Visible-Near-infrared absorbance data to changes in murine blood chemistry post infection. Spectral absorption results indicate a statistically relevant correlation at a 650 nm with infection for Plasmodium from between 4 and 7 sampling days' post infection, in spite of significant standard deviations among the sample populations for control and infected mice. No significant spectral absorption change for Trypanosoma infection was been detected from the current data. Corresponding stained slides of control and infected blood at each sampling date were taken with related infected cell counts determined and these correlate well for Plasmodium absorbance at 650 nm.

Fully automated method based on on-line molecularly imprinted polymer solid-phase extraction for determination of lovastatin in dietary supplements containing red yeast rice.

A fully automated method for the determination of lovastatin in dietary supplements containing red yeast rice has been developed. It uses a sequential injection analysis system combined with solid-phase extraction applying highly selective molecularly imprinted polymer sorbent. A miniaturized column for on-line extraction was prepared by packing 4.5 mg of the sorbent in a 5.0 × 2.5-mm-i.d. cartridge, which was used in the flow manifold. Sequential injection analysis manifold enabled all steps of lovastatin extraction and continuous spectrophotometric detection at 240 nm. A limit of detection of 60 µg g-1, a limit of quantitation of 200 µg g-1, and a linear calibration range of 200-2000 µg g-1 were achieved. Intra-day and inter-day precision values (RSD) were ≤ 6.7% and ≤ 4.9%, respectively, and method recovery values of spiked red yeast rice extracts at 200, 1000, and 2000 µg g-1 concentration levels were 82.9, 95.2, and 87.7%. Our method was used for determination of lovastatin lactone in four dietary supplements containing red yeast rice as a natural source of lovastatin, also known as monacolin K. The extracted samples were subsequently analyzed by the reference UHPLC-MS/MS method. Statistical comparison of results (F test, t test, α = 0.05) obtained by both methods did not reveal significant difference. A substantial advantage of the new automated approach is high sample throughput thanks to the analysis time of 7.5 min, miniaturization via down-scaling the extraction column, and smaller sample and solvent consumption, as well as reduced generation of waste. Graphical abstract ᅟ.

Validation of spectrophotometric determination of chlorogenic acid in fermentation broth and fruits.

A fast and accurate ultraviolet-visible (UV-vis) spectrophotometric method was established to determine the presence of chlorogenic acid (CGA) according to potassium ferricyanide-Fe (III) detection system. The reaction temperature and pH level greatly influenced the CGA absorption spectrum. Maximum UV-vis absorption wavelength occurred at 790 nm under the optimum conditions (at 25 °C and pH 7.0). The results of UV-vis were further assessed by comparing the results with those of high-performance liquid chromatography (HPLC). The proposed method showed a wide linear sensing range of 10.0-800.0 µg mL-1 (R2 = 0.9996) and a high degree of precision (%R.S.D. < 1.50) and recovery (%R.S.D. > 3.39). Results of CGA and HPLC methods correlated well with each other. The proposed accurate, rapid, sensitive, low-cost, and high-throughput method was successfully used to quantify CGA in fermentation broth and fruits (Mango). Therefore, it may be applied for measuring CGA in biological samples.

Determination of total and free ceftolozane and tazobactam in human plasma and interstitial fluid by HPLC-UV.

Ceftolozane/tazobactam is a new cephalosporin/beta-lactamase inhibitor combination. An HPLC-UV method is described for the determination of total and free ceftolozane and tazobactam in human plasma and in microdialysate of subcutaneous tissue, respectively. Separation was performed using a reversed-phase column with phosphate buffer/acetonitrile as eluent and photometric detection at 260 nm (ceftolozane) or 220 nm (tazobactam). Linearity has been shown down to ceftolozane/tazobactam 0.1/0.05 mg/L in plasma and 0.03/0.015 mg/L in saline, respectively. The plasma protein binding of both drugs as determined by ultrafiltration was less than 10%. Temperature, pH or relative centrifugation force (up to 3000 x g) had no significant impact on the protein binding. The method was applied to the determination of ceftolozane and tazobactam in plasma and interstitial fluid of healthy volunteers following intravenous infusion of ceftolozane/tazobactam 1.0/0.5 g.

Evaluating the performance of automated UV enzymatic assay for screening of glucose 6-phosphate dehydrogenase deficiency.

INTRODUCTION: A precise and reliable screening assay for glucose 6-phosphate dehydrogenase (G6PD) deficiency would greatly help avoiding unwanted outcomes due to bilirubin neurotoxicity in neonatal jaundice and antimalarial-induced haemolytic anaemia in malaria patients. Currently, available assays are laborious and require sophisticated laboratory expertise. This study aimed to evaluate the performance of a recently introduced automated screening assay for G6PD deficiency by comparing with a routine spectrophotometric assay. METHODS: An automated UV-based enzymatic (Mindray, PRC) and spectrophotometric assays were performed simultaneously in parallel to determine G6PD activity in 251 blood samples from the subjects. RESULTS: The median G6PD activity value from spectrophotometric assay was significantly lower than that of from the automated assay. The mean difference was -2.0 U/g haemoglobin (-7.3 to 3.2; P < 0.0001). The mean activity values of both assays were strongly correlated with Pearson's correlation coefficient of r = 0.8. Cohen's kappa statistics between assays was 0.77 (0.70-0.83). The sensitivity, specificity, positive and negative predictive values of the automated assay were 85.7%, 99.2%, 85.7%, 99.2%, respectively. The sensitivity and positive predictive values of the automated assay for identifying intermediate G6PD activity levels were 40.0% and 25.0%, respectively. Genotyping was performed to confirm G6PD deficient and intermediate samples. The turnaround time for 40 samples was 60 minutes for the automated assay and 300 minutes for spectrophotometric assay. CONCLUSION: The automated assay for the detection of G6PD deficiency is comparable to a routine spectrophotometric assay and help reducing sample handling time. However, the assay shows limitation in identifying individuals with G6PD intermediate.

Micellar liquid chromatographic method for the simultaneous determination of citalopram hydrobromide with its two demethylated metabolites. Utility as a diagnostic tool in forensic toxicology.

A micellar liquid chromatographic method has been developed for the simultaneous determination of citalopram hydrobromide (CTA) with its two demethylated metabolites namely; desmethyl citalopram hydrochloride (DCTA) and didesmethyl citalopram tartrate (DDCTA). Separation was conducted on a C18 column using a mobile phase composed of 0.18 M sodium dodecyl sulphate (SDS), 15% 1-propanol, 0.3% tri-ethylamine, adjusted to pH 4 with 0.2 M o-phosphoric acid and adopting UV detection at 240 nm. Analysis was performed at 60 °C applying a flow rate of 2 mL/min. The proposed method was linear over the concentration ranges of 1.0-200.0, 0.6-200.0, and 0.5-200.0 µg/mL for CTA, DCTA, and DDCTA respectively, with corresponding limits of detection (LOD) of 0.5, 0.4, and 0.3 µg/mL and limits of quantification (LOQ) of 0.8, 0.5, and 0.4 µg/mL. The method was fully validated which allowed its application for the determination of CTA in its tablets. Moreover, the proposed method was extended to assay CTA with its metabolites in rat tissue organs samples which allowed the method to be used as a diagnostic tool in forensic toxicology.

Molecular fingerprinting of nanoparticles in complex media with non-contact photoacoustics: beyond the light scattering limit.

Optical instruments can probe physical systems even to the level of individual molecules. In particular, every molecule, solution, and structure such as a living cell has a unique absorption spectrum representing a molecular fingerprint. This spectrum can help identify a particular molecule from others or quantify its concentration; however, scattering limits molecular fingerprinting within a complex compound and must be overcome. Here, we present a new, non-contact photoacoustic (PA)-based method that can almost completely remove the influence of background light scattering on absorption measurements in heterogeneous highly scattering solutions and, furthermore, separate the intrinsic absorption of nanoscale objects from their scattering. In particular, we measure pure absorption spectra for solutions of gold nanorods (GNRs) as an example of a plasmonic agent and show that these spectra differ from the extinction measured with conventional UV-VIS spectrophotometry. Finally, we show how the original GNR absorption changes when nanoparticles are internalized by cells.

Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS.

Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (∼1.5 µg).

Turn-off colorimetric sensor for sequence-specific recognition of single-stranded DNA based upon Y-shaped DNA structure.

A novel turn-off colorimetric sensor for sequence-specific recognition of single-stranded DNA (ssDNA) was established by combining Y-shaped DNA duplex and G-quadruplex-hemin DNAzyme. A G-rich single-stranded DNA (Oligo-1) displays peroxidase mimicking catalytic activity due to the specific binding with hemin in the presence of K+, which was able to catalyze the oxidation of colorless 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS2-) by H2O2 to generate green ABTS•- radical for colorimetric assay. Oligonucleotide 2 (Oligo-2) was partly complementary with Oligo-1 and the target DNA. Upon addition of target DNA, Oligo-1, Oligo-2 and target DNA can hybridize with each other to form Y-shaped DNA duplex. The DNAzyme sequence of Oligo-1 was partly caged into Y-shaped DNA duplex, resulting in the inactivation of the DNAzyme and a sharp decrease of the absorbance of the oxidation product of ABTS2-. Under the optimum condition, the absorbance decreased linearly with the concentration of target DNA over the range of 1.0-250 nM and the detection limit was 0.95 nM (3σ/slope) Moreover, satisfied result was obtained for the discrimination of single-base or two-base mismatched DNA.

Simultaneous quantitation of two direct acting hepatitis C antivirals (sofosbuvir and daclatasvir) by an HPLC-UV method designated for their pharmacokinetic study in rabbits.

Sofosbuvir (SOF) and daclatasvir (DCS) are novel, recently developed direct acting antiviral agents characterized by potent anti-hepatitis C virus action. A fast and efficient HPLC-UV method was developed, validated and applied for simultaneous determination of SOF and DCS in pharmaceutical formulations and biological fluids based on coupling liquid-liquid extraction with ultrasound and dual wavelength detection at λmax; 260 and 313 nm for SOF and DCS, respectively. This approach provided simple, sensitive, specific and cost-effective determination of the SOF-DCS mixture with good recoveries of the analytes from plasma. Analytes were separated within 7 min on C18 analytical column with acetonitrile-10 mM acetate buffer of pH 5.0 at a flow rate of 1.0 mL min-1. The linear ranges were 1-20 µg mL-1 for SOF and 0.6-6 µg mL-1 for DCS with correlation coefficients ≥0.9995. The detection limits in spiked rabbit plasma were 0.20 and 0.19 µg mL-1 for SOF and DCS, respectively. The method was validated according to ICH and US-FDA guidelines. Finally, the method was successfully applied for simultaneous pharmacokinetic studies of SOF and DCS in rabbits using rofecoxib as internal standard.

Multiwavelength UV-metric and pH-metric determination of the multiple dissociation constants of the lesinurad.

Potentiometric and spectrophotometric pH-titrations of the lesinurad for three consecutive dissociation constants determination were compared. Lesinurad is a selective inhibitor of uric acid reabsorption as part of a combination of medicines to treat high levels of uric acid in blood, also called hyperuricemia. Nonlinear regression of the pH-spectra with REACTLAB and SQUAD84 and of the pH-titration curve with ESAB determined three multiple close dissociation constants. The protonation scheme of lesinurad was suggested. A sparingly soluble anion L- of lesinurad was protonated to the still soluble species LH, LH2+ and LH32+ in pure water. Three consecutive thermodynamic dissociation constants were estimated pKTa1 = 2.09, pKTa2 = 4.25, pKTa3 = 6.58 at 25 °C and pKTa1 = 1.96, pKTa2 = 4.16, pKTa3 = 6.32 at 37 °C by UV-metric spectra analysis. The graph of molar absorption coefficients shows that the spectrum of species LH2+ and LH vary in colour, while protonation of chromophore LH2+ to LH32+ has less influence on chromophores in the lesinurad molecule. Three multiple thermodynamic dissociation constants of 1 × 10-4 M lesinurad were determined by the pH-metric analysis pKTa1 = 2.39, pKTa2 = 3.47, pKTa3 = 6.17 at 25 °C and pKTa1 = 2.08, pKTa2 = 3.29, pKTa3 = 6.03 at 37 °C. The values of enthalpy ΔH0(pKa1) = 19.19 kJ mol-1, ΔH0(pKa2) = 13.29 kJ mol-1, ΔH0(pKa3) = 38.39 kJ mol-1, show the dissociation process is endothermic. The positive values of ΔG0(pKa1) = 11.93 kJ mol-1, ΔG0(pKa2) = 24.26 kJ mol-1, ΔG0(pKa3) = 37.56 kJ mol-1 at 25 °C indicate that the dissociation process of pKa2 is not spontaneous, which was confirmed by its value of entropy ΔS0(pKa1) = 24.37 J mol-1, ΔS0(pKa2) = -36.79 J mol-1, ΔS0(pKa3) = 2.79 J mol-1. Three macro-dissociation constants of lesinurad and protonation locations were predicted by MARVIN and ACD/Percepta.

UV-vis Imaging of Piroxicam Supersaturation, Precipitation, and Dissolution in a Flow-Through Setup.

Evaluation of drug precipitation is important in order to address challenges regarding low and variable bioavailability of poorly water-soluble drugs, to assess potential risk of patient safety with infusion therapy, and to explore injectable in situ suspension-forming drug delivery systems. Generally, drug precipitation is assessed in vitro through solution concentration analysis methods. Dual-wavelength UV-vis imaging is a novel imaging technique that may provide an opportunity for simultaneously monitoring changes in both solution and solid phases during precipitation. In the present study, a multimodal approach integrating UV-vis imaging, light microscopy, and Raman spectroscopy was developed for characterization of piroxicam supersaturation, precipitation, and dissolution in a flow-through setup. A solution of piroxicam dissolved in 1-methyl-2-pyrrolidinone was injected into a flowing aqueous environment (pH 7.4), causing piroxicam to precipitate. Imaging at 405 and 280 nm monitored piroxicam concentration distributions during precipitation and revealed different supersaturation levels dependent on the initial concentration of the piroxicam solution. The combination with imaging at 525 nm, light microscopy, and Raman spectroscopy measurements demonstrated concentration-dependent precipitation and the formation, growth, and dissolution of individual particles. Results emphasize the importance of the specific hydrodynamic conditions on the piroxicam precipitation. The approach used may facilitate comprehensive understanding of drug precipitation and dissolution processes and may be developed further into a basic tool for formulation screening and development.

Determination of residual 4-nitrobenzaldehyde in chloramphenicol and its pharmaceutical formulation by HPLC with UV/Vis detection after derivatization with 3-nitrophenylhydrazine.

4-Nitrobenzaldehyde is the synthetic raw material and an important photodegradation product of chloramphenicol. With a structural "alert" of human genotoxic potential and reported mutagenicity, this compound should be controlled in drug substances as a potential genotoxic impurity. However, current analysis methods require complex pre-treatment processes and/or lack sufficient specificity and sensitivity. Nitrophenylhydrazine is a common carbonyl derivatization reagent used to determine the residual aromatic aldehydes in drug samples. In the present study, we report an unexpected advantage of 3-nitrophenylhydrazine hydrochloride as a derivatization reagent in the derivatization high-performance liquid chromatography-ultraviolet detection method to determine 4-nitrobenzaldehyde in chloramphenicol samples. Compared with other nitro-substituted phenylhydrazines, 3-nitrophenylhydrazine hydrochloride can minimize drug matrix and derivatization reagent interferences, since the maximum absorption wavelength of its derivative is significantly red-shifted to 397 nm. The derivatization conditions have been optimized in terms of reaction efficiency, including reaction temperature, time, and diluting solvent, through a design of experiments. As a result, after reaction with 500 µg mL-1 of 3-nitrophenylhydrazine hydrochloride in acetonitrile-water (70:30, v/v) at 60 °C for 30 min, the developed HPLC method could be used to determine 4-nitrobenzaldehyde with a limit of detection of 0.009 µg mL-1. The method was then validated and applied for the determination of residual 4-nitrobenzaldehyde in chloramphenicol and its eye-drop samples.