Multimaterial 3D Printed Fluidic Device for Measuring Pharmaceuticals in Biological Fluids.

Multimaterial 3D printing provides a unique capability for the creation of highly complex integrated devices where complementary functionality is realized using differences in material properties. Using a single and automated print process, microfluidic devices were fabricated containing (i) an optically transparent structure for fluorescence detection, (ii) electrodes for electrokinetic transport, (iii) a primary membrane to remove particulates and macromolecules including proteins, and (iv) a secondary membrane to concentrate small molecule targets. The device was used for the simultaneous extraction and concentration of small molecule pharmaceuticals from urine, which was followed by an on-chip electrophoretic separation of the concentrated targets for quantitative analysis. Owing to the high level of functional integration inside the device, manual handling was minimal and restricted to the introduction of the sample and buffer solutions. The 3D printed sample-in/answer-out device allowed the direct quantification of ampicillin-a small molecule pharmaceutical-in untreated urine within 3 min, down to 2 ppm. These results demonstrate the potential of 3D printing for on-demand fabrication of disposable, functionally integrated devices for low-cost point-of-collection (POC) diagnostics.

Multiplex detection of quality indicator molecule targets in urine using programmable hairpin probes based on a simple double-T type microchip electrophoresis platform and isothermal polymerase-catalyzed target recycling.

Recently, it has been crucial to be able to detect and quantify small molecular targets simultaneously in biological samples. Herein, a simple and conventional double-T type microchip electrophoresis (MCE) based platform for the multiplex detection of quality indicator molecule targets in urine, using ampicillin (AMPI), adenosine triphosphate (ATP) and estradiol (E2) as models, was developed. Several programmable hairpin probes (PHPs) were designed for detecting different targets and triggering isothermal polymerase-catalyzed target recycling (IPCTR) for signal amplification. Based on the target-responsive aptamer structure of PHP (Domain I), target recognition can induce PHP conformational transition and produce extension duplex DNA (dsDNA), assisted by primers & Bst polymerase. Afterwards, the target can be displaced to react with another PHP and initiate the next cycle. After several rounds of reaction, the dsDNA can be produced in large amounts by IPCTR. Three targets can be simultaneously converted to dsDNA fragments with different lengths, which can be separated and detected using MCE. Thus, a simple double-T type MCE based platform was successfully built for the homogeneous detection of multiplex targets in one channel. Under optimal conditions, the assay exhibited high throughput (48 samples per hour at most, not including reaction time) and sensitivity to three targets in urine with a detection limit of 1 nM (ATP), 0.05 nM (AMPI) and 0.1 nM (E2) respectively. The multiplex assay was successfully employed for the above three targets in several urine samples and combined the advantages of the high specificity of programmable hairpin probes, the excellent signal amplification of IPCTR, and the high through-put of MCE which can be employed for screening in biochemical analysis.

A simple method for the ampicillin determination in pharmaceuticals and human urine.

The new kinetically-based spectrophotometric method for the determination of microquantities of ampicillin is proposed in the present paper. Ampicillin degradation in strong alkaline medium was applied for the method development. The reaction rate was monitored at 265 nm. A differential variation of the tangent method was used to process the kinetic data. The method is valid over the 3.49-55.84 µg/mL ampicillin concentration interval with relative standard deviation (RSD) range 7.79-3.20%. The calculated detection limit was determined at 2.58 µg/mL based on the 3.3S0 criterion. The interference effects of some metal ions, anions, amino acids and other molecules were investigated in order to assess the method selectivity. The method was successfully applied to determining the content of ampicillin in commercial pharmaceutical preparations and human urine. The obtained results were in good correlation with the HPLC method results. The newly developed method is simple, inexpensive and efficient for the analysis of a large number of samples at room temperature in a short time.

[Simultaneous determination of sixteen antibiotics in human urine with ultra performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To develop a method for simultaneous determination of sixteen antibiotics in human urine by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: With Piperacillin as an internal standard, the target antibiotics in urine samples were enriched and purified by Oasis HLB solid phase extraction (SPE) cartridges, then separated in a ZORBAX SB-C18 column with a gradient elution of mobile phase of 0.1% formic acid water and acetonitrile, finally analyzed with multiple reaction monitoring (MRM) mode. RESULTS: The limits of detection (LOD) for these sixteen antibiotics were in the range of 0.05-10.0 ng/mL and the limits of quantification (LOQ) in the range of 0.25-20.0 ng/mL. Within the related linear range, the related coefficient (r) of sixteen antibiotics were all more than 0.995. Accuracies for these antibiotics were ranged from 82.0%-119.3%, the within-day precision were less than 13.9%. CONCLUSION: The developed method is sensitive, specific and appropriate for the analysis of antibiotics in forensic toxicology and therapeutic drugs monitoring.

Stable competitive enzyme-linked immunosorbent assay kit for rapid measurement of 11 active beta-lactams in milk, tissue, urine, and serum.

Beta-lactams are used as veterinary drugs for the treatment of food-producing animals. For consumer protection, legislation is in place to set limits for their residues. An enzyme-linked immunosorbent assay (ELISA) was developed which allowed, in a single reaction, the class-specific measurement of 11 beta-lactams, with limits of detection below European maximum residue limits. Determinations were feasible in milk, tissue, urine, and serum with simple and rapid sample preparation. In this format, the specific capture antibodies were precoated on the microtiter plate and horseradish peroxidase-labeled conjugate was used to compete with free beta-lactams. The stability of the precoated microtiter plate and conjugate was at least 1 year when stored at 2 to 8 degrees C; upon reconstitution, the conjugate was stable for 6 days at 2 to 8 degrees C. The stability of lyophilized ampicillin standards was at least 6 months when stored at 2 to 8 degrees C and at least 1 year when stored at -20 degrees C. A low cross-reactivity, 3.6%, was observed with ampicillin with open beta-lactam ring relative to 100% for intact ampicillin. Generic recognition was shown by relative cross-reactivity values ranging from 22 (penicillin V) to 144% (nafcillin). Cross-reactivity for cephalosporins was <0.1%. Intra- and interassay precisions expressed as coefficient of variation were typically 2-8%. The inhibitory concentration with 50% binding for ampicillin was typically 2 ppb. Recovery for different spiked levels was >70% with all the matrixes.

Significant Pharmacokinetic Interactions Between Quinine and Ampicillin-Cloxacillin Combination.

INTRODUCTION: The co-existence of malaria with bacterial infections is common in the tropics, hence the concurrent use of antimalarials and antibiotics. OBJECTIVE: This study aimed to investigate the effect on pharmacokinetics and antimicrobial activity of co-administration of quinine and combined ampicillin-cloxacillin. METHODS: In total, 14 healthy adults received single oral doses of ampicillin-cloxacillin combination alone and with quinine in a randomized crossover manner. Urine samples collected at predetermined intervals over 48 h were analysed. The effect of quinine on minimum inhibitory concentrations (MICs) of ampicillin and cloxacillin were determined against Staphylococcus aureus by agar diffusion, agar dilution, and broth dilution. RESULTS: Quinine significantly reduced the rate and extent of excretion of ampicillin and cloxacillin (p < 0.0002). The total amounts of ampicillin and cloxacillin excreted unchanged (Du(∞)) alone were 217.10 ± 53.82 and 199.0 ± 64.29 mg versus 126.40 ± 50.63 and 135.20 ± 52.24 mg, respectively, with quinine. Respective maximum excretion rates (dDu/dt max) for ampicillin and cloxacillin were 43.55 ± 19.41 and 77.64 ± 29.65 mg/h alone versus 18.01 ± 8.52 and 53.16 ± 20.72 mg/h with quinine. This indicates a significant reduction in Du(∞)and dDu/dt max by 41.78 and 58.65 % for ampicillin and 32.06 and 31.53 % for cloxacillin. Conversely, the disposition of quinine was unaffected by ampicillin-cloxacillin (p > 0.1). The MIC of antibiotics alone versus with quinine, respectively, were 0.11 ± 0.04 and 0.78 ± 0.1 µg/ml for ampicillin, and 0.18 ± 0.1 and 0.92 ± 0.4 µg/ml for cloxacillin, with a five- to sevenfold increase (p > 0.01); indicating a decrease in antimicrobial activity by quinine. CONCLUSIONS: Quinine therefore, reduced the bioavailability and the antimicrobial activity of ampicillin-cloxacillin upon co-administration, which may have therapeutic implications. Caution is required with the co-administration of these medicines.

Simultaneous determination of carvedilol and ampicillin sodium by first-derivative fluorometry in the presence of human serum albumin.

A sensitive and selective method for the simultaneous determination of carvedilol and ampicillin sodium (AS) in the presence of human serum albumin (HSA) is described. The maximum emission wavelengths of carvedilol and AS are at 357 nm and 426 nm with excitation at 254 nm, respectively. The first-derivative peaks of carvedilol and AS were at 337 nm and 398 nm, respectively. The linear-regression equations of the calibration graphs of carvedilol and AS were C = 0.0001H - 0.0063 and C = 1.530H - 43.84; the correlation coefficients were 0.9990 and 0.9986, respectively. The detection limits were 1 ng ml(-1) for carvedilol and 23 microg ml(-1) for AS, respectively. The effects of the pH, the stability of carvedilol and AS and foreign ions on the determination of carvedilol and AS were examined. The recoveries of carvedilol and AS were measured. This method is simple and can be used for the determination of carvedilol and AS in human serum and urine samples with satisfactory results.

Dose-dependent absorption of amoxycillin and bacampicillin.

The relationship between the relative absorption and increasing oral doses of amoxycillin and bacampicillin, a prodrug of ampicillin, was studied testing the hypothesis that a saturable transport system for aminopenicillins exists in the human gut. Each drug was given in four different doses in a randomized order to 12 fasting subjects. One group of subjects was given amoxycillin in single doses of 375, 750, 1500, and 3000 mg, while the other group received bacampicillin in 400, 800, 1600, and 3200 mg doses. The highest dose was four times larger than that normally used in clinical practice. Amoxycillin, and ampicillin generated from bacampicillin, were determined in plasma and urine by modern column liquid chromatographic methods. With increasing doses of the penicillins, there was a saturable increase in peak plasma concentration, plasma AUC, and urinary recovery. The mean (+/- SD) AUC values after 750, 1500, and 3000 mg amoxycillin were 86% +/- 13%, 70% +/- 16%, and 55% +/- 14% of that expected, when the expected ratio of AUC to dose was that of the 375 mg dose, assuming nonsaturable absorption. The corresponding AUC values after 800, 1600, and 3200 mg bacampicillin were 97% +/- 17%, 89% +/- 19%, and 76% +/- 11% of that expected from the results obtained after the 400 mg dose. The importance of dose of either drug for AUC and urinary recovery was analyzed according to a function implying capacity-limited absorption. The dose-dependency was most pronounced for amoxycillin (P less than 0.001). Renal drug clearance was stable within subjects throughout the dose range. Our results support the concept of capacity-limited absorption of aminopenicillins, probably by carrier-mediated transport. However, limited solubility of the compounds, especially of bacampicillin, may be a confounding factor.

Intrarenal concentration-time profile of ampicillin after administration of bacampicillin to patients before nephrectomy.

Nineteen patients with renal cancer who were to undergo nephrectomy received a single oral dose of bacampicillin 800 mg at approximately three (N = 5), six (N = 7), or nine (N = 7) hours before nephrectomy. Serum samples were taken at 30, 60, and 90 minutes after administration and at nephrectomy. Urine was collected immediately before operation. Samples of medulla and cortex tissues were immediately and carefully dissected from a healthy part of the removed kidney and homogenized with a buffer solution. All concentrations were determined by bioassay. Bacampicillin was well absorbed, with a mean +/- SD serum concentration of 16.0 +/- 11.4 mg/L at one hour. The concentrations in renal tissues were higher than the serum levels taken at the same time, and the highest concentrations were found in the urine. the mean ampicillin elimination half-life was approximately the same in the cortex, medulla, and urine, (2.7, 2.3, and 2.4 hr, respectively) but it was shorter in the serum (1.4 hr). Bacampicillin 800 mg produced concentrations in the renal tissues that were higher and more sustained than in the serum and were well above the minimum inhibitory concentrations for common urinary pathogens even ten hours after the dose.

High voltage electrophoresis of amino acids in urine containing ampicillin.

Analysis of amino acids is complicated by treatment with ampicillin. High voltage electrophoresis, which is convenient for the qualitative assessment of metabolic diseases, yields smears of ampicillin that mask the bands of citrulline, homocitrulline, phenylalanine, cystine, and homocystine. The addition of penicillinase prior to high voltage electrophoresis eliminates ampicillin and other penicillins and reveals these key amino acids.

Moment analysis for the separation of mean in vivo disintegration, dissolution, absorption, and disposition time of ampicillin products.

The in vivo disintegration, dissolution, absorption, and disposition processes of ampicillin products are separated by means of moment analysis. This method is model-independent, that is, any specific model is not assumed. The mean residence time (MRT), mean absorption time (MAT), mean dissolution time (MDT), and mean disintegration time (MDIT) are calculated for several dosage forms of ampicillin. The fraction of dose absorbed (F) is also separated into several fractions corresponding to these in vivo processes. Bioavailability and bioequivalence are discussed in terms of the zero and first moments. The flip-flop behavior of ampicillin is proved by the fact that the MRT following intravenous injection is less than the MAT of any oral dosage form. Absorption of released ampicillin is proved by the fact that the MRT following intravenous injection is less than the MAT of any oral dosage form. Absorption of released ampicillin is proved to be a rate-determining step, since the MRT of released ampicillin in the GI tract is the greatest of all MRT corresponding to the in vivo processes. Moment analysis is compared with classical compartment theory, and a new component concept is introduced.

Evaluation of the bioavailability of sarpicillin, the methoxymethyl ester of hetacillin, in humans.

The relative bioavailability of sarpicillin (the methoxymethyl ester of hetacillin) from three different oral dosage forms was compared in humans employing a three-way crossover study design. Each unit dose contained 250 mg of sarpicillin in terms of anhydrous ampicillin activity. The comparative bioavailability of a tablet containing added buffer, a liquid-filled capsule, and a standard powder-filled capsule was determined. The bioavailability parameters were Cmax, tmax, and AUC of intact plasma sarpicillin levels and saliva ampicillin levels. Significant correlation was found between plasma sarpicillin levels and saliva ampicillin levels following the administration of sarpicillin. All three formulations yielded statistically similar Cmax and AUC values with respect to plasma sarpicillin and saliva ampicillin levels. However, a more rapid absorption of intact sarpicillin was observed with the buffered tablet formulation, as reflected by significantly smaller tmax for both plasma sarpicillin and saliva ampicillin levels. The faster absorption from the tablet formulation gave more precise absorption among subjects.

Effect of obtrusive measures on antibiotic compliance.

The influence of compliance measurement activities on patient behavior was studied. The project measured the relationship among physical capsule counts, patient interviews, and the amounts of excreted ampicillin. The capsule counts and patient interviews were conducted in a manner that disguised their intent. Sixty college-age patients were assigned to one of three experimental groups: a telephone interview, a personal interview and capsule count, or a control group. Stimulation (interviews) occurred on the 2nd day of the prescribed regimen, and urine was collected on random days thereafter. Results indicated that both stimulation types were associated with more positive compliance rates. The influence diminished rapidly. The reactive influence of experimentor intervention associated with personal and phone communication was demonstrated.

Direct quantitative determination of optically active absorbing drugs in human urine by circular dichroism. Simultaneous direct determination of beta-lactam antibiotics and proteins.

Over 200 urine samples from 61 subjects were analyzed by circular dichroism spectroscopy. The results proved that this technique can be applied to the direct determination of optically active absorbing drugs in urine of subjects under multiple drug administration, independently of the presence of proteins, which can simultaneously be determined. A list of noninterfering drugs is included. The validity of the present method was confirmed by analysis of variance, the beta-lactam antibiotics ampicillin, cefoxitin, and cephalexin being chosen as model drugs and human albumin as the analytical standard for protein determination. The results demonstrated that the proposed method is accurate and precise, the correlation coefficients being higher than 0.9996. A circular dichroism and HPLC data comparison was successfully performed. The principal advantages of this method are simplicity, quickness, and economy, no derivatization or chromatographic separation steps being needed.

Pharmacokinetics of ampicillin and methoxymethyl ester of hetacillin in dogs.

Concentrations of ampicillin and the methoxymethyl ester of hetacillin were measured in plasma, prostatic fluid, and spinal fluid of dogs receiving ampicillin or the hetacillin ester by continuous intravenous infusion. The ratios of ampicillin in prostatic fluid relative to plasma levels were higher after dosing with the hetacillin ester. Pharmacokinetic parameter values were consistent with urinary excretion characteristics. Ready diffusion of the ester across biological membranes may facilitate eradication of pathogenic organisms in prostatic and spinal fluids.

Bioavailability of ampicillin and amoxicillin in fasted and nonfasted subjects.

The influence of various test meals and fluid volume on the relative bioavailability of ampicillin and amoxicillin was studied in healthy human subjects. Serum amoxicillin levels were somewhat, but not always, significantly higher than those of ampicillin from equivalent oral doses. Food ingested immediately before dosing reduced serum levels and urinary excretion of both antibiotics to a similar extent. Reduction of dosed water volume caused a marked decrease in serum amoxicillin levels in fasted subjects.

Pharmacokinetics of ampicillin during parenteral nutrition.

Seven healthy volunteers (four males, 20-28 years) were studied to determine the effect of parenteral nutrition (PN) on ampicillin clearance. Each volunteer received intravenous infusions of 1 liter of PN (3.75% amino acid and 10% dextrose) alternating with 1 liter of 10% dextrose (containing all additives as PN except for calcium and phosphorus); and a meal containing similar fluid volume, caloric, protein, and sodium content as the PN solution. Ampicillin (250 mg) was given intravenously 2 hr after commencement of each intravenous solution and 4 hr after the meal. During PN infusion, the mean (+/- SE) glomerular filtration rate (GFR) as indicated by creatinine clearance was 125 +/- 18 ml/min; ampicillin pharmacokinetic data included area under the serum ampicillin concentration-time curve of 899 +/- 118 micrograms min/ml, terminal elimination half life of 37 +/- 4.3 min, volume of distribution at steady state of 11.9 +/- 1.6 liter, total body clearance of 4.7 +/- 0.6 ml/min/kg, renal clearance of 3.8 +/- 0.5 ml/min/kg, and 82 +/- 6.7% of the ampicillin administered was excreted in urine over 10 hr. The results were not significantly altered by different nutrient regimens or the order of infusion of intravenous solutions. We conclude that the use of PN is unlikely to affect the pharmacokinetics of ampicillin provided the renal functions including GFR, remain unchanged.

A sensitive high performance liquid chromatographic assay of a new metabolite of ampicillin in man.

A sensitive, high-performance liquid chromatographic method has been developed for the determination of piperazine-2,5-dione, a new metabolite of ampicillin, in human urine. Piperazine-2,5-dione was separated from human urine on a C18-column using phosphate buffer-methanol (pH 3.5) as eluent. Subsequently, the effluent underwent a postcolumn reaction with mercurous chloride and was then detected at 305 nm. It was found that piperazine-2,5-dione was also excreted in the urine of a volunteer dosed with ampicillin.

Liquid chromatographic determination of ampicillin and its metabolites in human urine by postcolumn alkaline degradation.

A high-performance liquid chromatographic method has been developed for the determination of ampicillin (I) and its metabolites [5R,6R)-penicilloate (II), the (5S,6R)-epimer (III), and piperazine-2,5-dione (IV)) in human urine. The assay was based on the measurement of the absorbance at 300 nm following the postcolumn alkaline degradation with 0.75 M sodium hydroxide, 2 X 10(-3) M mercuric chloride, and 1 X 10(-2) M ethylenediaminetetraacetic acid disodium salt in solution. The limits of accurate determination were 0.5 microgram mL-1 for I, 2.0 microgram mL-1 for II and III, and 1.0 microgram mL-1 for IV in neat urine samples with a 10 microL injection. At concentrations of compounds I-IV of 5 micrograms mL-1, within- and between-run precisions were 1.10-4.03% and 0.93-2.34%, respectively. The urinary levels of I and its metabolites were quantified by the proposed method.

Fluorimetric determination of ampicillin by use of non-toxic catalysts. Estimation of beta-lactamase activity and parameters.

A fluorescence assay based on the use of biological reducing agents as catalysts rather than heavy metal ions has been developed for estimation of ampicillin concentrations. The assay is based on the conversion of ampicillin to its penicilloate, by treatment with sodium hydroxide, then neutralization, dilution with 0.5 M acetate buffer at pH4 and heating for 30 min at 100 degrees C in the presence of ascorbic acid (0.5 mg) and EDTA (50 microM). Reduced glutathione, cysteine and N-acetylcysteine also catalysed the development of fluorescence. A practical sensitivity range of 0.5-50 microM ampicillin was used. The assay was used to estimate ampicillin in some biological solutions to which the antibiotic has been added. Milk, blood serum, trypticase soy broth and nutrient broth containing 25 microM antibiotic assayed at 18.5, 21.7, 16.5 and 14.7 microM, respectively, with standard deviations between 1.2 and 0.7%. The low results were attributed to binding of some ampicillin by proteins or peptides which were removed by pretreatment. Urine containing 5 mM ampicillin assayed at 4.97 mM with a standard deviation of 3%. A modification of the procedure was used to measure beta-lactamase activity against ampicillin in several organisms in a fixed time assay. Kinetic parameters of a commercial beta-lactamase preparation from Bacillus cereus could also be determined by an additional modification. In both instances a correction was required for the intrinsic fluorescence of ampicillin remaining in the solution. The preparation examined had a Michaelis constant (Km) of 0.32 mM, a maximum velocity (Vmax) of 5398 micromol ampicillin hydrolysed mg(-1) min(-1), an apparent catalytic constant (Kcat, turnover number) of 20,220 s(-1) and a Kcat/Km ratio of 0.63 x 10(7) M(-1) s(-1). The major advantage of using this assay technique is that toxic metals are not used in the development of fluorescence so it is more environmentally acceptable. The technique is useful for examining beta-lactamase activity against ampicillin and might be useful for pharmaceutical products-for determining available therapeutic levels and for monitoring the activity of penicillin acylase against the penicilloate of ampicillin.