On-line Post-column Photochemical Derivatization Research Articles

Mycotoxin Detection in Maize, Commercial Feed, and Raw Dairy Milk Samples from Assiut City, Egypt.

This survey was conducted to investigate the contamination by multiple mycotoxins, aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), and zearalenone (ZEA) in 61 samples of maize and 17 commercial animal feed samples, and of aflatoxin M1 (AFM1) in raw dairy milk samples (n = 20) collected from Assiut City in Upper Egypt. Multi-mycotoxin immunoaffinity columns were used for samples cleanup and mycotoxin purification. An HPLC–FLD system with an on-line post-column photochemical derivatization was used for the detection of the target toxins. AFB1 was detected in both maize (n = 15) and feed (n = 8), with only one maize sample presenting a concentration above the maximum permissible level set by the Egyptian authorities. AFB2 was observed in six maize samples and in one feed sample, with a maximum value of 0.5 μg/kg. ZEA was detected only in feed samples (n = 4), with a maximum value of 3.5 μg/kg, while OTA, AFG1, and AFG2 were under the limits of detection. For milk, all the analyzed samples (100%) were contaminated with AFM1, and 14 samples (70%) presented concentrations above the maximum permissible level in the European Union (EU) (0.05 μg/kg). The concentrations ranged from 0.02 μg/kg to 0.19 μg/kg, except that of one sample, which was under the limit of quantification. The contamination rates in maize and animal feeds are not alarming. In contrast, the consumption of dairy milk samples in Assiut City may pose public health hazards, as AFM1 levels were found to exceed the international permissible limits. Further surveys are highly recommended in order to establish a database for mycotoxin occurrence in Egypt to minimize the possible health risks in animals and humans.

Affinity capture of aflatoxin B1 and B2 by aptamer-functionalized magnetic agarose microspheres prior to their determination by HPLC.

A novel adsorbent is described for magnetic solid-phase extraction (MSPE) of the aflatoxins AFB1 and AFB2 (AFBs). Magnetic agarose microspheres (MAMs) were functionalized with an aptamer to bind the AFBs which then were quantified by HPLC and on-line post-column photochemical derivatization with fluorescence detection. Streptavidin-conjugated MAMs were synthesized first by a highly reproducible strategy. They possess strong magnetism and high surface area. The MAMs were characterized by transmission electron microscopy, scanning electron microscopy, optical microscopy, laser diffraction particle size analyzer, Fourier transform infrared spectrometry, vibrating sample magnetometry and laser scanning confocal microscopy. Then, the AFB-aptamers were immobilized on MAMs through biotin-streptavidin interaction. Finally, the MSPE is performed by suspending the aptamer-modified MAMs in the sample. They are then collected by an external magnetic field and the AFBs are eluted with methanol/buffer (20:80). Several parameters affecting the coupling, capturing and eluting efficiency were optimized. Under the optimized conditions, the method is fast, has good linearity, high selectivity, and sensitivity. The LODs are 25pg·mL-1 for AFB1 and 10pg·mL-1 for AFB2. The binding capacity is 350 ± 8ng·g-1 for AFB1 and 384 ± 8ng·g-1 for AFB2, and the precision of the assay is <8%. The method was successfully applied to the analysis of AFBs in spiked maize samples. Graphical abstract Schematic of novel aptamer functionalized magnetic agarose microspheres (Apt-MAM) as magnetic adsorbents for simultaneous and specific affinity capture of aflatoxins B1 and B2 (AFBs).

Development of an Ultrasonication-Assisted Extraction Based HPLC With a Fluorescence Method for Sensitive Determination of Aflatoxins in Highly Acidic Hibiscus sabdariffa.

The high acidity and complex components of Hibiscus sabdariffa have provided major challenges for sensitive determination of trace aflatoxins. In this study, sample pretreatment of H. sabdariffa was systematically developed for sensitive high performance liquid chromatography-fluorescence detection (HPLC-FLD) after ultrasonication-assisted extraction, immunoaffinity column (IAC) clean-up and on-line post-column photochemical derivatization (PCD). Aflatoxins B1, B2, G1, G2 were extracted from samples by using methanol/water (70:30, v/v) with the addition of NaCl. The solutions were diluted 1:8 with 0.1 M phosphate buffer (pH 8.0) to negate the issues of high acidity and matrix interferences. The established method was validated with satisfactory linearity (R > 0.999), sensitivity (limits of detection (LODs) and limits of quantitation (LOQs) of 0.15–0.65 and 0.53–2.18 μg/kg, respectively), precision (RSD <11%), stability (RSD of 0.2–3.6%), and accuracy (recovery rates of 86.0–102.3%), which all met the stipulated analytical requirements. Analysis of 28 H. sabdariffa samples indicated that one sample incubated with Aspergillus flavus was positive with aflatoxin B1 (AFB1) at 3.11 μg/kg. The strategy developed in this study also has the potential to reliably extract and sensitively detect more mycotoxins in other complex acidic matrices, such as traditional Chinese medicines, foodstuffs, etc.

Method optimization and validation for the determination of eight sulfonamides in chicken muscle and eggs by modified QuEChERS and liquid chromatography with fluorescence detection

A simple, effective and reliable method for the determination of eight sulfonamide antibiotics (sulfadiazine, sulfapiridine, sulfamerazine, sulfamethazine, sulfachloropiridazine, sulfamethoxazole, sulfadoxine, sulfadimethoxin) in chicken muscle and eggs by liquid chromatography and fluorescence detection has been developed and validated. Sulfonamides do not present native fluorescence, however their direct determination was achieved by on-line post-column photochemical derivatization by UV irradiation. Sample treatment was based on QuEChERS with several modifications depending on the matrix. Egg extracts were cleaned-up using PSA for the dispersive solid phase extraction step. On the other hand, a new clean-up sorbent, Supel™ QuE Z-Sep+, has been successfully applied in chicken muscle extract and has proved to be effective for interference removal from this matrix. Under optimum conditions, recoveries from 65.9 to 88.1%, relative standard deviations lower than 10% (except for sulfachloropiridazine), and limits of quantification (LOQs) from 14 to 85μgkg−1 were achieved. Thus, the method complies with current European requirements.

Simultaneous determination of aflatoxins B₁, B₂, G₁, G₂ in Fructus Bruceae by high-performance liquid chromatography with online postcolumn photochemical derivatization.

A simple, reliable, and highly sensitive method for the simultaneous determination of aflatoxin B1 , B2 , G1 , G2 in Fructus Bruceae was developed using high-performance liquid chromatography coupled to online postcolumn photochemical derivatization and fluorescence detection. Aflatoxins were first extracted by a methanol/water mixture and then cleaned up with an AflaTest™ immunoaffinity column. Different clean-up and derivatization methods were compared and optimized. The established method was extensively validated to show satisfactory performance of linearity (R(2) ≥ 0.9997), recovery (74.3-100.8%), and precision (RSDs ≤ 2.8%) for the investigated aflatoxins. This proposed method was also applied to 11 F. Bruceae samples and the results showed that 10 out of 11 were contaminated with aflatoxins ranging from 0.26 to 27.52 μg/kg and the occurrence of aflatoxin B1 , the most toxic one, was as high as 91% in all the samples, highlighting the severe contamination and the necessity to set legal limits for aflatoxins in F. Bruceae.

Simultaneous determination of four aflatoxins and ochratoxin A in ginger and related products by HPLC with fluorescence detection after immunoaffinity column clean-up and postcolumn photochemical derivatization.

Ginger, a widely used spice and traditional Chinese medicine, is prone to be contaminated by mycotoxins. A simple, sensitive, and reproducible method based on immunoaffinity column clean-up coupled with HPLC and on-line postcolumn photochemical derivatization with fluorescence detection was developed for the simultaneous determination of aflatoxins (AFs) B1 , B2 , G1 , G2 , and ochratoxin A (OTA) in 25 batches of gingers and related products marketed in China for the first time. The samples were first extracted by ultrasonication with methanol/water (80:20, v/v) and then cleaned up with immunoaffinity columns for analysis. Under the optimized conditions, the LODs and LOQs for the five mycotoxins were 0.03-0.3 and 0.1-0.9 μg/kg, respectively. The average recoveries ranged from 81.3-100.8% for AFs and from 88.6-99.5% for OTA at three spiking levels. Good linearity was observed for the analytes with correlation coefficients all >0.9995. All moldy gingers were contaminated with at least one kind of the five investigated mycotoxins, while none of them were found in normal gingers. Ginger powder samples were contaminated slightly with the contamination levels below the LOQs, while ginger tea bags were mainly contaminated by OTA at 1.05-1.19μg/kg and ginger black tea bags were mainly contaminated by AFs at 3.37-5.76μg/kg. All the contamination levels were below the legally allowable limits.

Determination of aflatoxins in Nelumbinis Semen by immunoaffinity column clean-up and HPLC-FLD with on-line post-column photochemical derivatization and LC-MS/MS confirmation

To determine the contents of aflatoxin B1, B2, G1 and G2 in Nelumbinis Semen using on-line post-column photochemical derivatization-HPLC-FLD method and verify the method by LC-MS method. The samples were extracted with MeOH-H2O (80: 20) and purified with inmunoaffinity column, aflatoxins were analyzed by HPLC-FLD with post-column photochemical derivatizaton. The positive samples were further confirmed by LC-MS/MS. On optimum conditions, aflatoxin B1, G1 ranging 0.3-30 mg x L(-1) showed a good linear relationship with aflatoxin B2, G2 ranging 0.09-9.0 mg x L(-1) with r >0.999 9. The recoveries ranged between 86.7% and 99.1%, with RSDs all bellow 4.87%. LOD of aflatoxin B1, B2, G1 and G2 were 0.08, 0.03, 0.10, 0.03 microg x kg(-1), respectively. Among 20 Nelumbinis Semen samples, 14 were found to contain aflatoxin B1 ranging from 0.40 to 586 microg x kg(-1). The total content of aflatoxin B1, B2, G1 and G2 were between 0.40 and 602.5 microg x kg(-1). By LC-MS/MS method, the same fragment ions were founded in samples and the control group at the same retention times, ruling out the possibility of false positive samples. The method is simple, highly sensitive and reproducible for the determination of aflatoxins in Nelumbinis Semen.

Simultaneous determination of aflatoxin B1, B2, G1, G2 and ochratoxin A in Glycyrrhiza uralensis by HPLC-FLD after immunoaffinity column with online post-column photochemical derivatization

To develop a method for the simultaneous determination of aflatoxin B1, B2, G1, G2 and ochratoxin A in Glycyrrhiza uralensis by HPLC-FLD after immunoaffinity column with online post-column photochemical derivatization. Sample was extracted with MeOH: H2O (80:20) and cleaned up by immunoaffinity column. The toxins were separated by reversed-phase HPLC and the mobile phase was consisted of methanol and 0.5% acetic acid solution with gradient elution. The determination was carried out by fluorescence detector after photochemical derivatization. The detection limits of aflatoxin G2, G1, B2, B1 and ochratoxin A were 0.02, 0.06, 0.015, 0.03 and 0.25 microg x kg(-1), respectively. The recoveries of analytes were from 76.0% to 103% and the relative standard deviations (RSDs) were below 13%. The method is a simple, accurate and can be used to determine the contents of aflatoxin B1, B2, G1, G2 and ochratoxin A in G. uralensis simultaneously.

Bioanalytical method development for a generation 5 polyamidoamine folic acid methotrexate conjugated nanoparticle

Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9-N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 microL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.

Validation of a confirmatory analytical method for the determination of aflatoxins B1, B2, G1 and G2 in foods and feed materials by HPLC with on-line photochemical derivatization and fluorescence detection

A sensitive and selective analytical method for the simultaneous separation and quantitative determination of aflatoxins B1, B2, G1 and G2 in foodstuffs and materials for feed has been validated. The method is based on high performance liquid chromatography with on-line post-column photochemical derivatization and fluorescence detection. The chromatographic separation of aflatoxins was accomplished using a C18 column eluted with an isocratic mobile phase consisting of water, methanol and acetonitrile. The sample preparation required a simple extraction of aflatoxins with MeOH/H2O (80:20, v/v) and a purification step by immunoaffinity column cleanup. The total analysis time, including sample preparation and chromatographic separation, did not exceed 40 min with a run time of 10 min. The on-line photochemical derivatization ensures better results in terms of simplicity, sensitivity and reproducibility with respect to chemical derivatization techniques, and provides an increase of the peak resolution and an extent of automation in comparison with the electrochemical ones. The procedure for the determination of aflatoxins in food samples and cereals for animal consumption was extensively validated following Regulation (EC) No. 882/2004. Detection limits in wheat bran samples of 0.08 µg kg−1 for AFB1, 0.02 µg kg−1 for AFB2, 0.16 µg kg−1 for AFG1 and 0.04 µg kg−1 for AFG2 were attained. The method allows high recovery with mean values ranging from 72 to 94% and it satisfies the necessary requirements for sensitivity, linearity, selectivity, precision and ruggedness, demonstrating the conformity of the method with provisions of Regulation (EC) No. 401/2006.

Analysis of phenols in pharmaceuticals by liquid chromatography after pre-column labelling and on-line post-column photochemical derivatization

Reversed phase high-performance liquid chromatographic (RP-HPLC) methods are proposed for the analysis of phenol, thymol, chlorocresol and chloroxylenol in commercial pharmaceutical dosage forms. The use of 1-fluoro-2,4-dinitrobenzene as UV labelling reagent in pre-column derivatization has been investigated. The compound reacts rapidly (2min) under mild conditions (ambient temperature or 40°C) with phenols to give ethers that can be separated by RP-HPLC and detected at 292nm. The other procedure involves a post-column on-line photochemical conversion. A photoreactor was arranged between the analytical column and the fluorescence (λex=270nm, λem=310nm) and UV-diode array (λ=270nm) detector to enhance the performance of the methods. Additional informations of the analyte structure and photoreactivity by UV spectra (photoreactor ‘on’ and ‘off’) were obtained. The methods showed good selectivity and sensitivity.

On-line post-column photochemical derivatization in liquid chromatographic—diode-array detection analysis of binary drug mixtures

HPLC methods were developed for the analysis of pharmaceutical creams containing binary drug mixtures (betamethasone valerate—chlorocresol; hydrocortisone—miconazole nitrate; desonide pivalate—chlorhexidine; dexamethasone—clotrimazole; triamcinolone acetonide—econazole nitrate). The chromatographic separations were performed on C-18 and cyano columns under reversed-phase conditions. A post-column on-line photochemical reactor (irradiation at 254 nm) was arranged between the analytical column and the diode-array detector to enhance the performance of the method. Two UV spectra (photoreactor on and off) were obtained for each analyte and these additional sources of information proved to be useful for the unambiguous identification of the various analytes. The method was applied to the quality control of commercial creams using a solid-phase extraction procedure for the sample clean-up.

Application of high-performance liquid chromatography with diode-array detection and on-line post-column photochemical derivatization to the determination of analgesics

HPLC analyses of pharmaceutical dosage forms containing analgesics and related compounds (acetylsalicyclic acid, paracetamol, propyphenazone, caffeine and chlorpheniramine) were performed on C 18 and cyano columns under reversed-phase conditions. The performance of the methods was enhanced by introducing postcolumn on-line photochemical derivatization in combination with a diode-array detection. The column effluents were subjected on-line to UV irradiation (254 nm) and the characteristics photo-induced spectral modifications were useful for the unambiguous identification of the various analgesic compounds. The proposed HPLC methods were successfully applied to the analysis of commercially available analgesic dosage forms.

Simple and rapid determination of clomiphene cis and trans isomers in human plasma by high-performance liquid chromatography using on-line post-column photochemical derivatization and fluorescence detection

A validated, sensitive and rapid high-performance high-performance liquid chromatographic method has been developed for the analysis of both cis and trans isomers of clomiphene in human plasma. The method involves a new off-line pre-column solid-phase extraction for sample preparation of clomiphene from human plasma in the presence of an internal standard. Analysis was performed by isocratic elution on a LiChrospher 100 RP-18 5-μm column using on-line post-column photochemical derivatization, with fluorescence detection at 247 nm (excitation) and 378 nm (emission). The limit of quantitation was 0.75 ng/ml for the cis isomer and 1.25 ng/ml for the trans isomer. The precision and accuracy of method were between good laboratory practice (GLP) required limits.