Guanidine Additive Enabled Intermolecular ortho-Phthalaldehyde-Amine-Thiol Three-Component Reactions for Modular Constructions.

Recently, ortho-phthalaldehyde (OPA) is experiencing a renascence for the modification of proteins and peptides through OPA-amine two-component reactions for bioconjugation and intramolecular OPA-amine-thiol three-component reactions for cyclization. Historically, small thiol molecules were used in large excess to allow for the intermolecular OPA-amine-thiol reaction forming 1-thio-isoindole derivatives. In this study, we discovered that guanidine could serve as an effective additive to switch the intermolecular OPA-amine-thiol three-component reaction to a stoichiometric process and enable the modular construction of peptide-peptide, and peptide-drug conjugate structures. Thus, 12 model peptide-peptide conjugates have been synthesized from unprotected peptides featuring all proteinogenic residues. Besides, 6 peptide-drug conjugates have been prepared in one step, with excellent conversions and isolated yields. In addition, a conjugate product has been further functionalized by utilizing a premodified OPA derivative, demonstrating the versatility and flexibility of this reaction.

Highly selective fluorescence detection of Pt4+ over Pd2+ and Pt2+ using a polyethyleneimine-based nanosensor prepared via facile three-component reaction.

A novel polyethyleneimine (PEI)-based polymeric nanosensor (named PEIMP) was developed for specific fluorescence enhanced sensing of Pt4+ ion in aqueous media. The sensor was fabricated via "one-pot" three-component reaction using ortho-phthalaldehyde (OPA), PEI and mercaptopurine as raw materials, by which the formation of isoindole fluorophore and its chemical grafting onto PEI chain were achieved simultaneously. The morphology, size and structure of PEIMP have been characterized by various techniques. In buffered aqueous solution (pH 7.0), PEIMP had the ability to specifically bind with Pt4+ producing notable increase in fluorescence emission at 463 nm (excited at 395 nm). Based on investigations on the sensing mechanism, the fluorescence turn-on response towards Pt4+ was attributed to the binding of Pt4+ with purine group in PEIMP resulting in the inhibition of photoinduced electron transfer from purine to isoindole fluorophore. Under the optimal conditions (pH 7.0, incubated at 37 ℃ for 20 min) the detection of Pt4+ could be achieved with the linear range of 0.1-10 µM and the detection limit of 80 nM. The sensor had the advantages of low-cost raw materials, simple and environmental-friendly synthesis and analytical detection procedures. What's more, it could selectively and sensitively detect Pt4+ without the effects from common transition metal ions (Pb2+, Fe3+, Cr3+, Al3+, Ag+, Co2+, Hg2+, Cd2+, Cu2+, Mg2+, Ni2+, Mn2+, Zn2+), especially precious metalions of Pt2+ and Pd2+. The proposed method had been successfully applied to quantify Pt4+ in wastewater and urine samples, and also proved to be potential for monitoring Pt4+ in biological systems.

Characterization of Exposure to Cleaning Agents Among Health Workers in Two Southern African Tertiary Hospitals.

BACKGROUND: Whilst cleaning agents are commonly used in workplaces and homes, health workers (HWs) are at increased risk of exposure to significantly higher concentrations used to prevent healthcare-associated infections. Exposure assessment has been challenging partly because many are used simultaneously resulting in complex airborne exposures with various chemicals requiring different sampling techniques. The main objective of this study was to characterize exposures of HWs to various cleaning agents in two tertiary academic hospitals in Southern Africa. METHODS: A cross-sectional study of HWs was conducted in two tertiary hospitals in South Africa (SAH) and Tanzania (TAH). Exposure assessment involved systematic workplace observations, interviews with key personnel, passive personal environmental sampling for aldehydes (ortho-phthalaldehyde-OPA, glutaraldehyde and formaldehyde), and biomonitoring for chlorhexidine. RESULTS: Overall, 269 samples were collected from SAH, with 62 (23%) collected from HWs that used OPA on the day of monitoring. OPA was detectable in 6 (2%) of all samples analysed, all of which were collected in the gastrointestinal unit of the SAH. Overall, department, job title, individual HW use of OPA and duration of OPA use were the important predictors of OPA exposure. Formaldehyde was detectable in 103 (38%) samples (GM = 0.0025 ppm; range: <0.0030 to 0.0270). Formaldehyde levels were below the ACGIH TLV-TWA (0.1 ppm). While individual HW use and duration of formaldehyde use were not associated with formaldehyde exposure, working in an ear, nose, and throat ward was positively associated with detectable exposures (P-value = 0.002). Glutaraldehyde was not detected in samples from the SAH. In the preliminary sampling conducted in the TAH, glutaraldehyde was detectable in 8 (73%) of the 11 samples collected (GM = 0.003 ppm; range: <0.002 to 0.028). Glutaraldehyde levels were lower than the ACGIH's TLV-Ceiling Limit of 0.05 ppm. p-chloroaniline was detectable in 13 (4%) of the 336 urine samples (GM = 0.02 ng/ml range: <1.00 to 25.80). CONCLUSION: The study concluded that detectable exposures to OPA were isolated to certain departments and were dependent on the dedicated use of OPA by the HW being monitored. In contrast, low-level formaldehyde exposures were present throughout the hospital. There is a need for more sensitive exposure assessment techniques for chlorhexidine given its widespread use in the health sector.

Separation and detection of D-/L-serine by conventional HPLC.

D-serine has a role as an endogenous allosteric agonist of N-methyl-D-aspartate (NMDA) receptor in the mammalian brain. In this study, we present a detailed description of our method that measures D-/L-serine by using conventional high performance liquid chromatography (HPLC). • We reacted D-serine and L-serine with ortho-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) to form diastereomeric isoindole derivatives, then we separated and detected them by conventional reversed phase HPLC with electrochemical detector (ECD). • We present typical measurement data of rat brain homogenate as an example of a convenient, appropriate method for measuring brain concentrations of D-serine. • Since many peaks appear in biological samples, we confirmed that the peaks were derived from serine by treating the sample with D-amino oxidase and catalase to decompose D-serine. As a results, one peak disappeared, suggesting that it is derived from D-serine.

Development of a simple validated spectrofluorometric method for the assay of midodrine in tablets dosage form; Application to content uniformity testing.

A new sensitive spectrofluorimetric technique has been established for the estimation of midodrine hydrochloride. This method depends on the condensation reaction of ortho-phthalaldehyde with the primary aliphatic amine of midodrine in the presence of 2-mercapto-ethanol. The pH of the medium was adjusted to 9.0 using 0.1 M borate buffer. The fluorescence of the product was measured at wavelength of 451 nm after excitation at 334 nm. The method was rectilinear over a concentrations range of 0.1 to 1.5 µg mL-1. The lower detection and quantitation limits were 31 and 94 ng mL-1, respectively. The method was investigated following the guidelines of the International Council of Harmonization. Analysis of commercial tablet dosage form was carried out successfully by the current method with excellent recovery percentages and with no influence from coexisted pharmaceutical additives .This method was used to evaluate the uniformity of the contents of commercial tablets according to the United States Pharmacopoeia.

Evaluation of the ceiling levels of ortho-phthalaldehyde exposure among health care workers engaged in endoscope disinfection: A new methodology using video-exposure monitoring.

OBJECTIVES: The present study aimed to develop a method for measuring the ceiling level of ortho-phthalaldehyde (OPA) exposure and evaluate the ceiling levels of OPA exposure among health care workers who handle disinfectant solutions containing OPA for the disinfection of endoscopes. METHODS: The study consisted of a preliminary survey and main survey. In the preliminary survey, processes involving high-concentration exposure to OPA were identified by video-exposure monitoring (VEM). In the main survey, the ceiling levels of OPA exposure for high-concentration exposure processes identified from the results of the preliminary survey were determined using a measuring method combining sampling using a 2,4-dinitrophenylhydrazine-silica cartridge and analysis by high-performance liquid chromatography tandem mass spectrometry. RESULTS: In the preliminary survey, seven processes involving high-concentration exposure to OPA were identified by VEM. The duration of each process was short, lasting from 20 seconds to a few minutes. In the main survey, the OPA concentrations for the identified high-concentration exposure processes ranged from 1.18 to 4.49 ppb, which markedly exceeded the threshold limit value ceiling (TLV-C) of 0.1 ppb recommended by the American Conference of Governmental Industrial Hygienists. CONCLUSIONS: The method for measuring the ceiling level of OPA exposure was established using VEM and the highly sensitive method of chemical analysis; and we successfully evaluated the ceiling levels of OPA exposure among health care workers engaged in endoscope disinfection. This approach can also be applied to other chemical substances with recommended TLV-Cs, and important information for reducing exposure can thus be obtained.

Ortho-Phthalaldehyde (OPA)-based chemoselective protein bioconjugation and peptide cyclization.

Ortho-Phthalaldehyde (OPA)-amine reaction and OPA-amine-thiol reaction have been developed to effectively modify native peptides and proteins under the physiological conditions. First, OPA and its derivatives can rapidly and smoothly react with primary amine moieties in peptides and proteins to achieve native protein biconjugations. Furthermore, OPA-alkyne bifunctional linkers can be used for proteome profiling. Second, OPA-amine-thiol three-component reaction has been developed for chemoselective peptide cyclization, directly on unprotected peptides in the aqueous buffer. Moreover, this OPA-guided cyclic peptide can be further modified with the N-maleimide moiety in one pot to introduce additional functionalities. The development of this OPA based chemoselective bioconjugation and peptide cyclization extends the toolbox for protein chemical modification and construction of cyclic peptides.

FlICk (fluorescent isoindole crosslinking) for peptide stapling.

The rigidification of peptide secondary structure via stapling is an important and enduring goal in the development of functional peptides for biochemical and pharmaceutical applications. In addition, the incorporation of fluorophores and chromophores has been a sought-after application for creating peptidic probes of cellular function and localization. The combined application of peptide stapling and fluorescent-readout is featured by the reaction of ortho-phthalaldehydes to create isoindole staples, thus transforming inactive linear and monocyclic precursors into fluorescent or visibly colored monocyclic and bicyclic products with noted biological activity. Given its user-friendliness, we have termed this approach FlICk (fluorescent isoindole crosslink) chemistry and we have featured this application on an array of high-affinity macrocyclic α-MSH derivatives as well as for late-stage intra-annular isoindole stapling furnished a bicyclic peptide mimic of α-amanitin that is cytotoxic to CHO cells. The synthetic methods for preparing substituted ortho-phthalaldehydes along with subsequent applications to FlICk stapling are detailed herein.

Development of a measurement method to determine the ceiling exposure concentration of ortho-phthalaldehyde handling workers.

OBJECTIVES: The purpose of this research was to develop and validate an analytical method for rapid determination of the exposure of workers to ortho-phthalaldehyde (OPA) at the ceiling threshold concentration. METHODS: A 2,4-dinitrophenylhydrazine (DNPH)-silica cartridge was chosen as a sampler. OPA collected by the DNPH-silica cartridge was subsequently extracted with 5 mL of acetonitrile. A 50-µL aliquot of phosphoric acid/acetonitrile solution (2%, v/v) was added to 950 µL of the extraction solution and allowed to stand for 30 minutes at room temperature. This solution was then analyzed by high-performance liquid chromatography tandem mass spectrometry. The basic characteristics of the proposed method, such as recovery, repeatability, limit of quantification, and storage stability of the samples, were examined. RESULTS: The overall recoveries of OPA from OPA-spiked DNPH-silica cartridges were 93.6%-100.1% with relative standard deviations, representing the repeatability, of 1.5%-10.8%. The limit of quantification was 0.165 ng/sample. The recovery of OPA from DNPH-silica cartridges after 5 days of storage in a refrigerator exceeded 95%. CONCLUSIONS: The proposed method enabled the determination of the OPA concentration corresponding to the Threshold Limit Value-Ceiling of 0.1 ppb recommended by the American Conference of Governmental Industrial Hygienists, with a minimum sampling time of 18 seconds (corresponding to a sampling volume of 300 mL at 25°C and 1 atm). Thus, this method will be useful for estimating worker exposures to OPA.

Efficient synthesis of 4-substituted-ortho-phthalaldehyde analogues: toward the emergence of new building blocks.

4-Methoxy-ortho-phthalaldehyde and 4-hydroxy-ortho-phthalaldehyde are potentially useful molecules for fluorimetric analysis of a variety of amines and for the elaboration of complex molecular architectures. Nevertheless, literature generally describes their synthesis in very low yield (below 5%), mainly due to the inefficiency of the last oxidation step. In this paper, we report a reliable synthesis of 4-substituted-ortho-phthalaldehyde analogues in 51% overall yield owing to the addition of a protecting step of the unstable key intermediate 4,5-dihydroisobenzofuran-5-ol. Oxidation and deprotection steps were also studied in order to provide an effective availability of these two dialdehyde compounds that may increase their future applications.

Comparison of disinfection effects of three disinfectants on gastroscope:a meta analysis / 中华消化内镜杂志

Objective To evaluate the disinfect effects of glutaraldehyde, ortho-phthalaldehyde ( OPA ) and peracetic acid on gastroscopy disinfection. Methods Relevant literature from PubMed, Cochrane Library, web of science, Embase, CNKI, CBM, VIP were retrieved to collect the randomized controlled trials on disinfection by glutaraldehyde, OPA and peracetic acid on gastroscope. Literature was selected according to the inclusion and exclusion criteria. The RevMan 5. 3. 4. 0 statistic software was used to extract data and a meta-analysis was performed. Results A total of 18 RCT were included. There were significant differences in the disinfect effects between the OPA group and the glutaraldehyde group ( OR=2. 02, 95％CI:1. 88-1. 27, P<0. 00001), and between the peracetic acid group and the glutaraldehyde group ( OR = 2. 79, 95％CI:1. 52-5. 11, P = 0. 0009 ) . There were no significant differences in the disinfection effect between the OPA group and peracetic acid group ( OR=1. 30,95％CI:0. 62-2. 73, P=0. 49) . Conclusion The disinfect effects of OPA and peracetic acid are similar, which are superior to glutaraldehyde. Compared with OPA and glutaraldehyde, peracetic acid is a better choice considering its good disinfect effect and low cost.

Differentiating between Testicular Toxicity and Sexual Immaturity in Ortho-phthalaldehyde Inhalation Toxicity Studies in Rats and Mice.

Early deaths of young or juvenile animals (before sexual maturation is achieved) in routine regulatory safety studies present pathologists and toxicologists with the challenge of interpreting findings in the male reproductive tract. Additionally, the advent of toxicity testing regulations has resulted in a growing need for the use of juvenile animals in toxicology studies. Here, we present the reproductive toxicity findings from a 13-week inhalation toxicity study with ortho-phthalaldehyde (OPA) in male rats and mice as a case example for working through this challenging task. In this study with OPA, survival was significantly reduced in the two highest exposure concentrations of OPA tested. Early deaths and histopathological lesions in the testes and epididymides were generally also limited to these two highest exposure groups. Therefore, there was concern that peripubertal morphological features could be a confounding factor for the histopathological evaluation of exposure-related testicular and epididymal findings. Although it can be difficult to differentiate exposure-related effects from the normal morphological features defining peripubertal changes in the testes and epididymides in animals that die early in a toxicity study, the use of age-matched controls in this case study with OPA provided a reference and aided in the differentiation of these effects.

Evaluation of the respiratory tract toxicity of ortho-phthalaldehyde, a proposed alternative for the chemical disinfectant glutaraldehyde.

ortho-Phthalaldehyde (OPA) is a high-level chemical disinfectant that is commonly used for chemical sterilization of dental and medical instruments as an alternative to glutaraldehyde, a known skin and respiratory sensitizer. Concern for safe levels of human exposure remains due to a lack of toxicity data as well as human case reports of skin and respiratory sensitization following OPA exposure. The present study evaluated the inhalational toxicity of OPA in Harlan Sprague-Dawley rats and B6C3F1/N mice. Groups of 10 male and female rats and mice were exposed to OPA by whole-body inhalation for 3 months at concentrations of 0 (control), 0.44, 0.88, 1.75, 3.5, or 7.0 ppm. Rats and mice developed a spectrum of lesions at sites of contact throughout the respiratory tract (nose, larynx, trachea, lung), as well as in the skin and eye, consistent with a severe irritant response. In general, histologic lesions (necrosis, inflammation, regeneration, hyperplasia and metaplasia) occurred at deeper sites within the respiratory tract with increasing exposure concentration. As a first site of contact, the nose exhibited the greatest response to OPA exposure and resulted in an increased incidence, severity and variety of lesions compared to a previous study of glutaraldehyde exposure at similar exposure concentrations. This increased response in the nasal cavity, combined with extensive lesions throughout the respiratory tract, provides concern for use of OPA as a replacement for glutaraldehyde as a high-level disinfectant.

[Anaphylaxis and allergic reactions during surgery and medical procedures]. / Anafilaxias y reacciones alérgicas durante cirugías y procedimientos médicos.

Anaphylaxis during anesthesia is an unpredictable, severe, and rare reaction. It has an incidence of 1/10 000 to 1/20 000 surgeries. In most series, the responsible drugs include neuromuscular blocking agents, latex, or antibiotics. The frequency and etiology of systemic allergic reactions in other medical procedures are largely unknown. The identification of responsible drugs of anaphylaxis is a complex task, requiring testing of all medications and substances used during surgery. We describe our experience in a retrospective study of 15 patients. Ten subjects developed anaphylaxis during surgery, two in endoscopic studies and one in a trans-vaginal ultrasound. The remaining two subjects, one in a trans-vaginal ultrasound and another during a dental procedure had a systemic allergic reaction. We studied all patients with all medications administered during the procedures, including latex and detergents and disinfectants. Three surgeries had to be suspended at induction of anesthesia, five were stopped incomplete and two were completed. Both patients that presented a reaction during endoscopy required intensive care unit admission and the rest were observed in a Hospital. The responsible drugs during surgery anaphylaxis were neuromuscular blocking agents, latex, patent blue, and ranitidine. Ortho-phthalaldehyde (OPA) was identified during endoscopic studies; latex was responsible in transvaginal ultrasounds; and amoxicillin in the dental procedure. The aim of the present article is to review our experience studying allergic systemic reactions and anaphylaxis during general anesthesia and medical procedures, emphasizing the severity of these reactions and the need for causative drug identification.

Anafilaxias y reacciones alérgicas durante cirugías y procedimientos médicos / Anaphylaxis and allergic reactions during surgery and medical procedures

Las reacciones anafilácticas intraoperatorias son impredecibles, infrecuentes y pueden poner en riesgo al paciente. Tienen una incidencia de 1/10 000 a 1/20 000 produciéndose en la mayoría de los casos por bloqueantes musculares, látex y antibióticos. No hay estadística de las reacciones alérgicas sistémicas durante otros procedimientos médicos. El estudio diagnóstico posterior a una reacción es complejo debiendo incluir toda la medicación utilizada en el procedimiento. En este estudio retrospectivo describimos 15 pacientes, de los cuales 10 tuvieron reacciones anafilácticas en un procedimiento quirúrgico, 2 en endoscopías y 1 en una ecografía transvaginal. Los dos pacientes restantes presentaron una reacción alérgica sistémica durante una ecografía transvaginal y un procedimiento odontológico. Estudiamos los pacientes con toda la medicación utilizada, incluimos látex y, eventualmente, los detergentes y desinfectantes, de haber sido empleados. Tres de las 10 cirugías no pudieron realizarse por desarrollarse la reacción durante la inducción anestésica, en cinco casos debieron interrumpirse y solo en dos se terminaron. Las reacciones posteriores a endoscopías fueron severas, requiriendo internación en terapia intensiva; las reacciones en ecografías transvaginales y procedimientos odontológicos fueron asistidas en emergencias. Los agentes causales en las cirugías incluyeron bloqueantes musculares, látex, cefalosporina, azul patente y ranitidina; en endoscopías el agente causal fue el orto-ftalaldehído (OPA), en las ecografías transvaginales el látex y en el procedimiento odontológico la amoxicilina. El objetivo de este artículo es describir la etiología de las reacciones alérgicas sistémicas y anafilácticas intraoperatorias y en procedimientos médicos, recalcando su gravedad y la necesidad de su identificación.

Anaphylaxis during anesthesia is an unpredictable, severe, and rare reaction. It has an incidence of 1/10 000 to 1/20 000 surgeries. In most series, the responsible drugs include neuromuscular blocking agents, latex, or antibiotics. The frequency and etiology of systemic allergic reactions in other medical procedures are largely unknown. The identification of responsible drugs of anaphylaxis is a complex task, requiring testing of all medications and substances used during surgery. We describe our experience in a retrospective study of 15 patients. Ten subjects developed anaphylaxis during surgery, two in endoscopic studies and one in a trans-vaginal ultrasound. The remaining two subjects, one in a trans-vaginal ultrasound and another during a dental procedure had a systemic allergic reaction. We studied all patients with all medications administered during the procedures, including latex and detergents and disinfectants. Three surgeries had to be suspended at induction of anesthesia, five were stopped incomplete and two were completed. Both patients that presented a reaction during endoscopy required intensive care unit admission and the rest were observed in a Hospital. The responsible drugs during surgery anaphylaxis were neuromuscular blocking agents, latex, patent blue, and ranitidine. Ortho-phthalaldehyde (OPA) was identified during endoscopic studies; latex was responsible in transvaginal ultrasounds; and amoxicillin in the dental procedure. The aim of the present article is to review our experience studying allergic systemic reactions and anaphylaxis during general anesthesia and medical procedures, emphasizing the severity of these reactions and the need for causative drug identification.

Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells.

Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40µM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples.

Comparison of efficacy and cost-effectiveness of 0.55% ortho-phthalaldehyde and 2% glutaraldehyde for disinfection of laryngoscopes: A prospective pilot study.

BACKGROUND AND AIMS: The laryngoscope is a potential source of cross-infection as it involves contact with the mucous membrane, saliva and occasionally blood. This study compared efficacy and cost-effectiveness of two Centre for Disease Control approved agents for disinfection of laryngoscope blades. METHODS: One hundred and sixty patients requiring laryngoscopy and intubation for general anaesthesia were randomly allocated into two groups. After tracheal intubation, used laryngoscope blades were cleaned with tap water. The blades were then immersed in either 2% w/v glutaraldehyde for a contact time of 20 min or 0.55% w/v ortho-phthalaldehyde (OPA) for 10 min. The handles were wiped with 0.5% w/v chlorhexidine wipes. Samples were collected using sterile cotton swabs from the tip, flange and light bulb area of the laryngoscope blade and one from the handle. They were cultured aerobically on blood and McConkey agar. RESULTS: In 2% glutaraldehyde group, of 240 samples sent from the blades, 2 (0.8%) showed the growth of methicillin-resistant coagulase-negative staphylococci (MRCONS) and Enterobacter. In OPA group, of 240 samples, 2 (0.8%) showed growth of MRCONS. Thus, 2% glutaraldehyde and 0.55% OPA were comparable in terms of efficacy of disinfection. Growth was seen on 4 out of 160 handles. CONCLUSIONS: We suggest OPA for high-level disinfection of laryngoscope blades as it is equally efficacious as compared to glutaraldehyde, with a shorter contact time and available as a ready to use formulation.

An HPLC method for the determination of selected amino acids in human embryo culture medium.

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP18e or Ascentis Express C18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.

High-performance liquid chromatography evaluation of the enantiomeric purity of amino acids by means of automated precolumn derivatization with ortho-phthalaldehyde and chiral thiols.

The use of ortho-phthalaldehyde (OPA) for the derivatization of amino acids (AA) is well known. It enables the separation of the derivatives on common reversed phase columns and improves the sensitivity with fluorescence detection. With the use of a chiral thiol an indirect enantioseparation of chiral amines and AAs is feasible. The major drawback of the OPA-derivatization is the poor stability of the products. Here, a method with an in-needle derivatization procedure is optimized to facilitate a quantitative conversion of the AA with OPA and the chiral thiols N-acetyl-L-cysteine or N-isobutyryl-L-cysteine, followed by a subsequent analysis, eluding the stability issue. Both enantiomers of a single AA were separated as OPA-derivatives with a pentafluorophenyl column and a gradient program consisting of 50 mM sodium acetate buffer pH = 5.0 and acetonitrile. Fluorescence detection is commonly used to achieve sufficient sensitivity. In this study, the enantiomeric impurity of an AA can be detected indirectly with common UV spectrophotometric detection with a limit of quantitation of 0.04%. Seventeen different L-AAs were tested and the amount of D-AA for each individual AA was calculated by means of area normalization, which ranged from not detectable up to 4.29%. The recovery of the minor enantiomer of L- and D-AA was demonstrated for three AAs at a 0.04% level and ranged between 92.3 and 113.3%, with the relative standard deviation between 1.7 and 8.2%.