In vitro monitoring sub-nanogram amounts analgin in human urine by its inhibitory of the luminol-periodate chemiluminescence reaction using reagent immobilization release technique

A selective and sensitive as well as rapid chemiluminescence (CL) flow sensor for the determination of analgin is described. The analytical reagents involved in chemiluminescence reaction, luminol and periodate, were both immobilized on an anion-exchange column. The CL signals produced by the reaction between luminol and periodate, which were eluted from the column through water injection, were decreased in the presence of analgin. Analgin was sensed by measuring the decrement of CL intensity, and which was observed linear over the logarithm of analgin concentration range of 0.1 to 50.0 ng mL(-1), and the limit of detection was 0.04 ng mL(-1) (3ó). At a flow rate of 2.0 mL min(-1), including sampling and washing, the detection could be performed in 0.5 min with a relative standard deviation of less than 3.0%. The proposed procedure was applied successfully in the monitoring of analgin in human urine samples without any pre-treatment process. It was found that the analgin concentration reached its maximum after being orally administrated for 4 h, and the analgin metabolism ratio in 10 h was 9.28% in the body of volunteers. The flow sensor offered reagentless procedures and remarkable stability in determination of analgin, and could be easily reused over 80 h.     

Flow-injection chemiluminescence determination of catecholamines based on their enhancing effects on the luminol-potassium periodate system.

A rapid and sensitive chemiluminescence (CL) method using flow injection analysis is described for the determination of four catecholamines, dopamine, adrenaline, isoprenaline and noradrenaline, based on their greatly enhancing effects on the CL reaction of luminol-potassium periodate in basic solutions. The optimized chemical conditions for the chemiluminescence reaction were 1.0 x 10(-4) mol/L luminol and 1.0 x 10(-5) mol/L potassium periodate in 0.2 mol/L sodium hydroxide (NaOH). Under the optimized conditions, the calibration graphs relating the CL signal intensity (peak height) to the concentration of the analytes were curvilinear and they were suitable for determining dopamine, adrenaline, isoprenaline, and noradrenaline in the range 0.1-10 ng/mL, 0.1-100 ng/mL, 1-100 ng/mL and 5-50 ng/mL, respectively, with the relative standard deviations of 0.8-1.7%. The detection limits of the method are 0.02 ng/mL for dopamine, 0.01 ng/mL for adrenaline, 0.1 ng/mL for isoprenaline and 2.0 ng/mL for noradrenaline. The sampling frequency was calculated to be about 60/h. The selectivity of the method was good, because a series of common ions or excipients, such as K(+), Ba(2+), CO(3)(2-), NO(3)(-), SO(4)(2-), PO(4)(3-), sodium citrate, sodium bisulphite, oxidate dopamine, starch, lactose, carbamide and gelatin, could not produce interference when their concentrations were 1000-fold than those of dopamine. The present method was successfully applied to the determination of the four catecholamines in pharmaceutical injections.     

Determination of puerarin in biological samples and its application to a pharmacokinetic study by flow‐injection chemiluminescence

A simple and sensitive flow injection–chemiluminescence (FI–CL) method has been developed for the determination of puerarin, based on the fact that puerarin can greatly inhibit CL of the luminol–H₂O₂–haemoglobin system. The inhibition of CL intensity was linear to the logarithm of the concentration of puerarin in the range 0.08–10.0 μg/mL (r² = 0.9912). The limit of detection was 0.05 μg/mL (3σ) and the relative standard deviation (RSD) for 1.0 μg/mL (n = 11) of puerarin solution was 1.4%. Coupled with solid‐phase extraction (SPE) as the sample pretreatment, the determination of puerarin in biological samples and a preliminary pharmocokinetic study of puerarin in rats were performed. The recoveries for plasma and urine at three different concentrations were 89.2–110.0% and 91.4–104.8%, respectively. The pharmacokinetics of puerarin in plasma of rat coincides with the two‐compartment open model. The T_1/2α, T_1/2β, CL/F, V_Z/F, AUC_{(0 – t)}, MRT_{(0 – ∞)}, T_max and C_max were 0.77 ± 0.21 h, 7.55 ± 2.64 h, 2.43 ± 1.02 L/kg/h, 11.40 ± 3.45 L/kg, 56.67 ± 10.65 mg/h/L, 5.04 ± 2.78 h, 1.00 ± 0.35 h and 19.70 ± 4.67 μg/mL, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of puerarin in rat plasma by rapid resolution liquid chromatography tandem mass spectrometry in positive ionization mode

A highly sensitive and specific method of rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) in positive ionization mode has been developed and validated for pharmacokinetic study of puerarin in rat plasma. Chromatography was carried out on a Zorbax XDB C18 reversed-phase column using a mobile phase comprising a mixture of methanol and 0.05% acetic acid in water (35:65, v/v) with a flow rate of 0.3 mL/min from 0 min to 5.4 min and then 0.6 mL/min from 5.41 min to 12 min. The mass spectrometer operated in ESI positive ionization mode. Multiple reaction monitoring (MRM) was used to measure puerarin and tectoridin (internal standard). The method was sensitive with a detection limit of 0.33 ng/mL. A good linear response was observed over a range of 10-2000 ng/mL in rat plasma. The inter- and intra-day precision ranged from 2.97% to 7.52% and accuracy from 93.70% to 101.60%. This validated method was applied successfully to a pharmacokinetic study in rat plasma after intravenous administration of puerarin. The main pharmacokinetic parameters were as follows: AUC(0→t) 45.37±13.19 (mgh/L), AUC(0→∞) 47.03±14.78 (mgh/L), MRT 1.03±0.46 (h), T(1/2) 1.31±0.31 (h), V(ss) 0.09±0.02 (L), V(z) 0.17±0.04 (L), Cl 0.10±0.04 (L/h).     

Ultrasensitive assay of clindamycin in medicine and bio-fluids with chemiluminescence detection.

A simple chemiluminescence (CL) method using flow injection has been developed for the determination of clindamycin, based on the inhibitory effect of clindamycin on the CL generated from the luminol-K(3)Fe(CN)(6) system in alkaline medium. It was found that the decrement of CL intensity was linear with the logarithm of clindamycin concentration over the range 0.7-1000 ng/mL. The detection limit was 0.2 ng/mL with a relative standard deviation (RSD) of <5.0% (n = 7). At a flow rate of 3.0 mL/min, a complete analytical process could be performed within 0.5 min, including sampling and washing. The proposed procedure was applied successfully to the determination of clindamycin in pharmaceutical preparations and human urine without pretreatment.     

A novel green analytical procedure for monitoring of azoxystrobin in water samples by a flow injection chemiluminescence method with off‐line ultrasonic treatment

A simple and green flow injection chemiluminescence (FI‐CL) method for determination of the fungicide azoxystrobin was described for the first time. CL signal was generated when azoxystrobin was injected into a mixed stream of luminol and KMnO₄. The CL signal of azoxystrobin could be greatly improved when an off‐line ultrasonic treatment was adopted. Meanwhile, the signal intensity increases with the analyte concentration proportionally. Several variables, such as the ultrasonic parameters, flow rate of reagents, concentrations of sodium hydroxide solution and CL reagents (potassium permanganate, luminol) were investigated, and the optimal CL conditions were obtained. Under optimal conditions, the linear range of 1–100 ng/mL for azoxystrobin was obtained and the detection limit (3σ) was determined as 0.13 ng/mL. The relative standard deviation was 1.5% for 10 consecutive measurements of 20 ng/mL azoxystrobin. The method has been applied to the determination of azoxystrobin residues in water samples. Copyright © 2012 John Wiley & Sons, Ltd.     

One-step rapid determination and purification of puerarin from Radix puerariae by n-octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monolith

n-Octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monoliths were prepared for rapid screening, determination and one-step purification of puerarin from Radix puerariae (a crude extract of the root of Pueraria lobata). The modified monolith showed a specific surface area of 17.8 m(2) g(-1), an average pore size of 0.76 microm and a total porosity of 60.8%. Fast separation of R. puerariae crude extract was achieved within 5 min at a flow velocity of 722 cm h(-1) resulting in a puerarin purity of 97%, with a recovery of 85%. This demonstrates the potential of n-octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monolith for the rapid analysis and separation of isoflavonoids. Preparative scale sample loading (12 mg in 2 mL) resulted in a purity of 95%, and a recovery of about 69%. HPLC, FTIR, MS and (1)H NMR spectroscopy were used for the characterization and quantification of puerarin in isolated fraction.     

Flow injection analysis of riboflavin with chemiluminescence detection using a N-halo compounds-luminol system.

A chemiluminescent method for the determination of riboflavin is described. The method is based on the chemiluminescence (CL) generated during the oxidation of luminol by N-bromosuccinimide (NBS) and N-chlorosuccinimide (NCS) in alkaline medium. It was found that riboflavin could greatly enhance this CL intensity when present in the luminol solution. Based on this observation, a new flow-injection CL method for the determination of riboflavin is proposed in this paper. The detection limits were 7.5 ng/mL and 3.5 ng/mL of riboflavin for the NBS- and NCS-luminol CL systems, respectively. The relative CL intensity was linear, with riboflavin concentration in the range 19-600 ng/mL and 600-2000 ng/mL for the NBS-luminol CL system, and 12-200 ng/mL and 200-2000 ng/mL for the NCS-luminol CL system. The results obtained for the assay of pharmaceutical preparations compared well with those obtained by the official method and demonstrated good accuracy and precision.     

Flow-injection determination of carbaryl and carbofuran based on KMnO(4)-Na(2)SO(3) chemiluminescence detection.

A flow-injection method is described for the determination of carbaryl and carbofuran. It was found that a strong chemiluminescence (CL) signal was generated when these pesticides were mixed with Na(2)SO(3) and KMnO(4) in acidic medium. Under the optimum experimental conditions, the enhanced CL intensity was linear, with the concentrations in the range 0.1-2.0 microg/mL (r(2) = 0.9996 and 0.9993, n = 6) with relative standard deviation (n = 4) in the range 1.0-2.3%. The limits of detection (3sigma blank) were 10 and 50 ng/mL, respectively, with a sample throughput of 180/h. The proposed method was applied to determine carbaryl and carbofuran in freshwaters with satisfactory results. Most metal and non-metal ions and some pesticides, such as carbophenothion and aldicarb, do not interfere with the determination. Dinoseb, diazinon and malathion calibration graphs (in the range 0.2-2.0 microg/mL, r(2) = 0.9966-0.9988, n = 6) were also established with relative standard deviations (n = 4) in the range 1.2-2.0% with limits of detection (3sigma blank) in the range 100-300 ng/mL.     

Flow‐injection chemiluminescence method to detect a β2 adrenergic agonist

A new method for the detection of β₂ adrenergic agonists was developed based on the chemiluminescence (CL) reaction of β₂ adrenergic agonist with potassium ferricyanide–luminol CL. The effect of β₂ adrenergic agonists including isoprenaline hydrochloride, salbutamol sulfate, terbutaline sulfate and ractopamine on the CL intensity of potassium ferricyanide–luminol was discovered. Detection of the β₂ adrenergic agonist was carried out in a flow system. Using uniform design experimentation, the influence factors of CL were optimized. The optimal experimental conditions were 1 mmol/L of potassium ferricyanide, 10 µmol/L of luminol, 1.2 mmol/L of sodium hydroxide, a flow speed of 2.6 mL/min and a distance of 1.2 cm from ‘Y₂’ to the flow cell. The linear ranges and limit of detection were 10–100 and 5 ng/mL for isoprenaline hydrochloride, 20–100 and 5 ng/mL for salbutamol sulfate, 8–200 and 1 ng/mL for terbutaline sulfate, 20–100 and 4 ng/mL for ractopamine, respectively. The proposed method allowed 200 injections/h with excellent repeatability and precision. It was successfully applied to the determination of three β₂ adrenergic agonists in commercial pharmaceutical formulations with recoveries in the range of 96.8–98.5%. The possible CL reaction mechanism of potassium ferricyanide–luminol–β₂ adrenergic agonist was discussed from the UV/vis spectra. Copyright © 2014 John Wiley & Sons, Ltd.     

Interovarian relationship in the secretion of progesterone during the luteal phase of the capuchin monkey (Cebus apella)

In basal conditions, progesterone concentrations were similar in the ovarian veins of the ovary +CL (3211 +/- 526 ng/ml) and the ovary -CL (3165 +/- 554 ng/ml), but after blocking the blood flow between the ovary +CL and the uterus, the progesterone values in the vein draining the ovary -CL decreased to 1218 +/- 394 ng/ml (P less than 0.01). When [3H]progesterone was injected in the ovary +CL, the radioactivity appeared earlier and more concentrated in the vein draining the ovary -CL (30 sec, 0.53% of injected dose) than in the femoral vein (150 sec, 0.08% of injected dose). Removal of the ovary +CL was followed by a brief maintenance of peripheral progesterone within luteal-phase levels. The in-vitro progesterone production by a suspension of cells isolated from the corpus luteum was 47.5 +/- 12.8 ng/ml/2 h, whereas luteal-like cells isolated from the ovary -CL secreted 14.3 +/- 6.0 ng/ml/2 h (P less than 0.01) into the medium. We therefore suggest that the symmetrical and high secretion rate of progesterone by the ovaries of the capuchin monkey indicates a between-ovary communication system, and that the luteal-like tissue of the ovary -CL can produce relatively large amounts of progesterone.     

Quantitative determination of bilirubin by inhibition of chemiluminescence from lucigenin.

Bilirubin is a metabolic breakdown product of blood haem, of great biological and diagnostic importance. A new chemiluminescence (CL) method has been developed for the quantification of bilirubin. The method is combined with the flow injection analysis (FIA) technique and based on the inhibition effect of bilirubin on the CL from the lucigenin-hydrogen peroxide system in an alkaline medium. Under the optimum conditions, the decreased CL intensity was proportional to the concentration of bilirubin, in the range 0.0585-58.47 microg/mL. The detection limit estimated from the calibration graph was about 7.8826 ng/mL. The relative standard deviation (RSD) of 10 parallel measurements (1 x 10(-4) mol/L bilirubin) was 2.5%. Recoveries of bilirubin were found to fall in the range 94-97.5% using control sera. The method is interference-free, fast and easy to carry out.     

Effects of pretreatment with 8018 on the toxicokinetics of soman in rabbits and distribution in mice

The effects of 8018 [3-(2'-phenyl-2'-cyclopentyl-2'-hydroxyl-ethoxy)quinuclidine] on the elimination of soman in rabbits blood and distribution in mice brain and diaphragm were investigated using the chirasil capillary gas chromatographic analysis method. In all experiments, the concentration of P(+)soman was below the detection limit (<0.1 ng x mL(-1)). 8018 (1 mg x kg(-1), im, 10 min pre-treated) could significantly reduce the concentration of P(-)soman in rabbit blood from 53.6 +/- 13.3 to 26.2 +/- 9.70 ng x mL(-1) blood as compared to soman-treated control animal at 15 s following soman injection (43.2 microg x kg(-1), iv). Toxicokinetic parameters showed 8018 could increase clearance (CL((S))) from 20.8 +/- 1.54 to 38.2 +/- 15.3 mLx kg(-1) x s(-1) and reduce AUC of P(-)soman from 2.08 +/- 0.151 to 1.30 +/- 0.564 mg x s x L(-1). 8018 could reduce the concentration P(-)soman in diaphragm from 74.7, 70.5, 88.7 ng x g(-1) to 54.5 45.6, 50.0 ng x g(-1) at the time of 30, 90, 120 s after intoxication of soman subcutaneously vs. soman control respectively, but it had no influence on the concentration of free P(-)soman in brain. Isotope trace experiments showed that it could significantly increase the distribution amount of bound [3H]soman in mice plasma and small intestine during 0-120 min after mice received [3H]soman (0.544 GBq.119 microg x kg(-1), sc) compared to soman control group.     

Flow‐injection chemiluminescence and electrogenerated chemiluminescence determination of escitalopram oxalate in tablet form

Rapid, simple and highly sensitive flow‐injection (FI) chemiluminescence (CL) and flow‐injection electrogenerated chemiluminescence (ECL) methods were developed for the determination of escitalopram oxalate (ESC), a selective serotonin reuptake inhibitor used as an antidepressant drug. The CL method was based on the CL reaction of ESC with acidic cerium(IV) and tris(2,2'‐bipyridyl)ruthenium(II) (Ru). Various experimental parameters affecting CL intensity were carefully studied and optimised. The method enabled the determination of 0.001‐50 µg/mL of ESC in bulk form with a correlation coefficient r = 0.9999. The limit of detection (LOD) was 0.01 ng/mL (S/N = 3). The ECL method was based on the ECL reaction of Ru with the drug in an acidic medium, permitting the determination of ESC in the range of 0.00001‐70 µg/mL with r = 0.9999 and LOD of 1 x 10^‐4 ng/mL. The proposed methods were applied to the determination of ESC in commercial tablets. The results were compared statistically with those obtained from a published method using t‐ and F‐tests. Copyright © 2012 John Wiley & Sons, Ltd.     

Flow injection chemiluminescence determination of dihydralazine sulphate based on permanganate oxidation sensitized by rhodamine B.

A novel flow injection chemiluminescence (CL) method for the determination of dihydralazine sulphate (DHZS) is described. The method is based on the CL produced during the oxidation of DHZS by acidic permanganate solution in the presence of rhodamine B. Rhodamine B is suggested as a fluorescing compound for the energy-transferred excitation. The CL emission allows quantitation of DHZS concentration in the range 5-800 ng/mL, with a detection limit of 1.9 ng/mL (3sigma). The experimental conditions for the CL reaction are optimized and the possible reaction mechanism is discussed. The method has been applied to the determination of DHZS in pharmaceutical preparations and compares well with the high performance liquid chromatography (HPLC) method.     

Premature luteal regression in goats superovulated with PMSG: effect of hCG or GnRH administration during the early luteal phase

Twenty-two goats were superovulated with PMSG; 84 h after the onset of estrus the goats were treated with saline solution (control group n = 7), hCG (hCG group, n = 7), or GnRH (GnRH group, n = 8). The ovaries of all the goats were laparoscopically examined 3 and 6 d after the onset of estrus. In each case the CL were counted and classified according to their appearance as normal-looking or as regressing. Blood samples for progesterone determination were collected every 12 h from Day 1 to Day 6. Premature luteal regression was considered to have occurred if progesterone concentrations declined to less than 1 ng/mL by Day 6. According to progesterone concentrations, 57.5, 0 and 37.5% of the goats underwent premature luteal regression in the control, hCG and GnRH groups, respectively. Progesterone concentrations were higher in the hCG group than in the other groups on Days 5 and 6 post estrus (P < 0.05). The control group was the only one in which there was a significant (P < 0.05) increase in the number of regressing CL between Day 3 (1.6 +/- 1.4) and Day 6 (7.3 +/- 1.4). It was also the only group in which there was a significant decrease in the number of normal-looking CL between Day 3 (12.6 +/- 2.1) and Day 6 (2.6 +/- 2.1). On Day 6 the animals treated with hCG had significantly more normal-looking CL (12.0 +/- 2.3) than those in the control group (2.6 +/- 2.1). The number of large follicles present on the ovaries on Day 6 post estrus had negative correlations with progesterone concentrations (P = 0.05) and with the number of normal-looking CL (P < 0.05). It is concluded that the administration of hCG 84 h after the onset of estrus prevents premature luteal regression in goats superovulated with PMSG.     

Agreement between ultrasonographic classification of the CL and plasma progesterone concentration in dairy cows

We evaluated the agreement between ultrasonographic characteristics of the corpus luteum (CL) and plasma progesterone (P4) concentration in dairy cows. In Phase I of the study, the ovaries of 8 cows were ultrasonographically examined, and P4 was analyzed daily from estrus (Day 0) to Day 4, then at Day 7 and Day 10, and again daily from Day 17 to the onset of next estrus. In Phase 2, the ovaries of 157 randomly selected Friesian cows were examined once by ultrasonography, and blood samples collected concurrently were analyzed for plasma P4. On the basis of the P4 values, the function of CLs was classified as follows: 1) non-secretory CL when plasma P4 was lower than 1 ng/mL (n=41); 2) evolving CL when plasma P4 was between 1 and 4 ng/mL (n=55); and 3) mid-cycle CL when plasma P4 was more than 4 ng/mL (n=61). On the basis of ultrasonographic examination, 3 additional groups were established (absence of CL, evolving CL, midcycle CL). Ultrasonographic characteristics and size of Day 3 to 4 CLs and their respective plasma P4 concentrations were not distinguishable from those of CLs observed 3 to 4 d before the subsequent estrus. The degree of agreement between the two classification was 72%. The data indicate that the functional classification of CLs is difficult to determine based on ultrasonography alone.     

Local changes in blood flow within the early and midcycle corpus luteum after prostaglandin F(2 alpha) injection in the cow

One of the postulated main luteolytic actions of prostaglandin (PG) F(2 alpha) is to decrease ovarian blood flow. However, before Day 5 of the normal cycle, the corpus luteum (CL) is refractory to the luteolytic action of PGF(2 alpha). Therefore, we aimed to determine in detail the real-time changes in intraluteal blood flow after PGF(2 alpha) injection at the early and middle stages of the estrous cycle in the cow. Normally cycling cows at Day 4 (early CL, n = 5) or Days 10--12 (mid CL, n = 5) of the estrous cycle (estrus = Day 0) were examined by transrectal color and pulsed Doppler ultrasonography to determine the blood flow area, the time-averaged maximum velocity (TAMXV), and the volume of the CL after an i.m. injection of a PGF(2 alpha) analogue. Ultrasonographic examinations were carried out just before PG injection (0 h) and then at 0.5, 1, 2, 4, 8, 12, 24, and 48 h after the injection. Blood samples were collected at each of these times for progesterone (P) determination. The ratio of the colored area to a sectional plane at the maximum diameter of the CL was used as a quantitative index of the changes in blood flow within the luteal tissue. Blood flow within the midcycle CL initially increased (P < 0.05) at 0.5-2 h, decreased at 4 h to the same levels observed at 0 h, and then further decreased to a lower level from 8 h (P < 0.05) to 48 h (P < 0.001). Plasma P concentrations decreased (P < 0.05) from 4.7 +/- 0.5 ng/ml (0 h) to 0.6 +/- 0.2 ng/ml (24 h). The TAMXV and CL volume decreased at 8 h (P < 0.05) and further decreased (P < 0.001) from 12 to 24 h after PG injection, indicating structural luteolysis. These changes were not detected in the early CL, in which luteolysis did not occur. In the early CL, the blood flow gradually increased in parallel with the CL volume, plasma P concentration, and TAMXV from Day 4 to Day 6. The present results indicate that PGF(2 alpha) induces an acute blood flow increase followed by a decrease in the midcycle CL but not in the early CL. This transitory increase may trigger the luteolytic cascade. The lack of intraluteal vascular response to PG injection in the early CL appears to be directly correlated with the ability to be resistant to PG.     

Sensitive determination of synephrine by flow-injection chemiluminescence.

It was found that light emission produced by the oxidation of luminol by potassium ferricyanide in basic medium was enhanced by synephrine, an anti-obesity drug. The optimum conditions for this chemiluminescent reaction were studied in detail in a flow injection system and employed in a new, simple and rapid method for the determination of synephrine. A mechanism for this reaction is proposed, based on the chemiluminescence reaction spectra. In the optimum conditions, CL intensity is proportional to concentration of synephrine in the 0.008-1 microg/mL range. The limit of detection is 1.6 ng/mL for synephrine (3sigma), and the relative standard deviation (n = 11) is 2.6% for 0.5 microg/mL synephrine. The method was applied to the determination of synephrine in herbal products, citrus fruit and biological fluids. The recoveries were satisfactory (90-102%). The results given by the proposed method are in good agreement with those given by HPLC-UV.