Acoustic-resonance spectrometry as a process analytical technology for the quantification of active pharmaceutical ingredient in semi-solids

The purpose of this study was to demonstrate acoustic resonance spectrometry (ARS) as an alternative process analytical technology to near infrared (NIR) spectroscopy for the quantification of active pharmaceutical ingradient (API) in semi-solids such as creams, gels, ointments, and lotions. The ARS used for this research was an inexpensive instrument constructed from readily available parts. Acoustic-resonance spectra were collected with a frequency spectrum from 0 to 22.05 KHz. NIR data were collected from 1100 to 2500 nm. Using 1-point net analyte signal (NAS) calibration, NIR for the API (colloidal oatmeal [CO]) gave anr ² prediction accuracy of 0.971, and a standard error of performance (SEP) of 0.517%CO. ARS for the API resulted in anr ² of 0.983 and SEP of 0.317%CO. NAS calibration is compared with principal component regression. This research demonstrates that ARS can sometimes outperform NIR spectrometry and can be an effective analytical method for the quantification of API in semi-solids. ARS requires no sample preparation, provides larger penetration depths into lotions than optical techniques, and measures API concentrations faster and more accurately. These results suggest that ARS is a useful process analytical technology (PAT). Published: July 14, 2006     

Quantitation of drug content in a low dosage formulation by transmission near infrared spectroscopy

A transmission near infrared (NIR) spectroscopic method has been developed for the nondestructive determination of drug content in tablets with less than 1% weight of active ingredient per weight of formulation (m/m) drug content. Tablets were manufactured with drug concentrations of ∼0.5%, 0.7%, and 1.0% (m/m) and ranging in drug content from 0.71 to 2.51 mg per tablet. Transmission NIR spectra were obtained for 110 tablets that constituted the training set for the calibration model developed with partial least squares regression. The reference method for the calibration model was a validated UV spectrophotometric method. Several data preprocessing methods were used to reduce the effect of scattering on the NIR spectra and base the calibration model on spectral changes related to the drug concentration changes. The final calibration model included the spectral range from 11 216 to 8662 cm⁻¹ the standard normal variate (SNV), and first derivative spectral pretreatments. This model was used to predict an independent set of 48 tablets with a root mean standard error of prediction (RMSEP) of 0.14 mg, and a bias of only −0.05 mg per tablet. The study showed that transmission NIR spectroscopy is a viable alternative for nondestructive testing of low drug content tablets, available for the analysis of large numbers of tablets during process development and as a tool to detect drug agglomeration and evaluate process improvement efforts. Published: March 24, 2006     

Once-daily tablet formulation and in vitro release evaluation of cefpodoxime using hydroxypropyl methylcellulose: A technical note

Summary and Conclusions  Decreasing the dose frequency of cefpodoxime proxetil increases patient compliance; patients prefer to take the drug once daily. It also improves the rate of bacterial killing and hastens the cure from the indications, and therefore increases compliance. The hydrophilic matrix of HPMC controlled the cefpodoxime proxetil release effectively for 24 hours; hence, the formulation can be considered as a once-daily sustained-release tablet of cefpodoxime proxetil. The formulation showed acceptable pharmacotechnical properties and assay requirements. In vitro dissolution studies indicated a sustained-release pattern throughout 24 hours of the study that was comparable to the theoretical release profile. Drug release kinetics indicated that drug release was best explained by Higuchi’s equation, as these plots showed the highest linearity (r ²=0.9734), but a close relationship was also noted with zero-order kinetics (r ²=0.9708). Korsmeyer’s plots indicated ann value of 0.57, which was indicative of an anomalous diffusion mechanism or diffusion coupled with erosion; hence, the drug release was controlled by more than one process. Hixson-Crowell plots indicated a change in surface area and diameter of the tablets with the progressive dissolution of the matrix as a function of time. Published: September 22, 2006     

Coronary angiography and percutaneous transluminal coronary angioplasty procedures: Evaluation of patients' maximum skin dose using Gafchromic films and a comparison of local levels with reference levels proposed in the literature

The aim of this study was to evaluate the maximum skin dose (MSD) in patients undergoing interventional cardiology procedures, obtaining local reference levels and comparing these with the reference levels proposed in the literature.The patients' MSD was measured using Gafchromic XR type R films. In order to evaluate reference levels, the number of images acquired, the fluoroscopy times and the KAP_TOTAL were recorded for each procedure.For the evaluation of the MSD, 8 coronary angiography (CA) and 16 percutaneous transluminal coronary angioplasty (PTCA) procedures, carried out in the period from May to June 2008, were analyzed.For the CA procedures the MSD values were below 0.5 Gy.For the PTCA procedures, we found a fairly good correlation between fluoroscopy time and MSD (r = 0.80, p = 0.0002) and between MSD and WFP (r = 0.863, p < 0.0001); there was a strong correlation between KAP_TOTAL and MSD (r = 0.904, p < 0.0001). Since the correlation between KAP_TOTAL and MSD is more striking than that with fluoroscopic time and the WFP, KAP measurements are suitable for online skin dosimetry and may, therefore, be used to avoid radiation-induced skin injuries. A MSD greater than 3 Gy occurred in only one procedure.For calculus of the local reference levels, we extended the data-gathering to 30 procedure CA and to 40 PTCA: we compared local practice with that in other centers using the guidance levels proposed by Balter et al. Our median KAP values were below these proposed guidance levels; our mean KAP values were above these proposed action levels. From a first application of the proposed reference levels, it appears that, according to the recommendations of Balter et al. an investigation into local practice is not necessary.     

Individual microflora beget unique oral microcosms

AIMS: To examine the efficacy of the multiple Sorbarod device (MSD) for the reproduction of inter-individual variations in oral microbiotas. The MSD supports sessile growth on parallel cellulose filters, perfused with artificial saliva. This enables biofilms (BF) to be grown and sampled, together with released cells in eluted medium (perfusates, PAs). METHODS AND RESULTS: Two sets of triplicate MSDs were established. One set was inoculated using fresh saliva from three separate volunteers; the second set was inoculated from one saliva donor. Both were incubated in an anaerobic cabinet. BF and PA were analysed at 24-h intervals by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Hierarchical dendrograms were constructed in order to sort community fingerprints over time, based on community relatedness. The MSD supported complex oral communities, as evidenced by DGGE (>20 distinct DGGE bands) and confocal scanning laser microscopy. DGGE band sequencing revealed bacteriological diversity and a high incidence of anaerobic species, including Prevotella sp. Dendrograms demonstrated marked inter-individual variation in the relative species abundance within salivary inocula from different volunteers (DV) and each associated MSD (all >45%, majority c. 85% concordance). Less variation was shown between triplicate models established using saliva from a single volunteer (SV) (all >58%; majority c. 95% concordance). PAs clustered together with the associated biofilms and inocula in the majority of cases for the DV MSDs whilst SV MSD community profiles clustered between replicate MSDs. CONCLUSIONS: Data indicate that marked inter-individual variations in human salivary composition can be partially replicated in individualized MSD microcosms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the in vitro reproduction of individual oral microbiotas and suggests that taking inter-individual variability into account will increase the relevance of microcosm studies.     

Feasibility of setting up generic alert levels for maximum skin dose in fluoroscopically guided procedures

PurposeThe feasibility of setting-up generic, hospital-independent dose alert levels to initiate vigilance on possible skin injuries in interventional procedures was studied for three high-dose procedures (chemoembolization (TACE) of the liver, neuro-embolization (NE) and percutaneous coronary intervention (PCI)) in 9 European countries.MethodsGafchromic® films and thermoluminescent dosimeters (TLD) were used to determine the Maximum Skin Dose (MSD). Correlation of the online dose indicators (fluoroscopy time, kerma- or dose-area product (KAP or DAP) and cumulative air kerma at interventional reference point (K_a,r)) with MSD was evaluated and used to establish the alert levels corresponding to a MSD of 2 Gy and 5 Gy. The uncertainties of alert levels in terms of DAP and K_a,r, and uncertainty of MSD were calculated.ResultsAbout 20–30% of all MSD values exceeded 2 Gy while only 2–6% exceeded 5 Gy. The correlations suggest that both DAP and K_a,r can be used as a dose indicator for alert levels (Pearson correlation coefficient p mostly >0.8), while fluoroscopy time is not suitable (p mostly <0.6). Generic alert levels based on DAP (Gy cm²) were suggested for MSD of both 2 Gy and 5 Gy (for 5 Gy: TACE 750, PCI 250 and NE 400). The suggested levels are close to the lowest values published in several other studies. The uncertainty of the MSD was estimated to be around 10–15% and of hospital-specific skin dose alert levels about 20–30% (with coverage factor k = 1).ConclusionsThe generic alert levels are feasible for some cases but should be used with caution, only as the first approximation, while hospital-specific alert levels are preferred as the final approach.     

Nondestructive and on-line monitoring of tablets using light-induced fluorescence technology

A system using light-induced fluorescence (LIF) technology was developed for rapid and nondestructive analysis of active pharmaceutical ingredients on tablet surfaces. Nonhomogenous tablets with defined layer of active ingredients were made by 3-Dimensional Printing technology to determine penetration depths of the light source and the resultant fluorescence responses. The LIF method of analysis showed penetration to depths of up to 3 mm into tablets. A correlation between LIF signals from analysis of tablet surfaces and the total drug content of the respective tablets was established. This method of surface analysis was verified with UV spectrometric methods for the total drug content of each respective tablet. The results from a small sample population of tablets made from both homogeneous and nonhomogeneous powder mixtures established good correlation between LIF surface monitoring and total tablet content. The use of on-line monitoring of the individual tablet for surface content demonstrated consistent LIF profiles from simulated production rates up to 3000 tablets a minute. The instrument was also field tested successfully on a tablet analyzer.     

Mechanical Property Characterization of Bilayered Tablets using Nondestructive Air-Coupled Acoustics

A noncontact/nondestructive air-coupled acoustic technique to be potentially used in mechanical property determination of bilayer tablets is presented. In the reported experiments, a bilayer tablet is vibrated via an acoustic field of an air-coupled transducer in a frequency range sufficiently high to excite several vibrational modes (harmonics) of the tablet. The tablet vibrational transient responses at a number of measurement points on the tablet are acquired by a laser vibrometer in a noncontact manner. An iterative computational procedure based on the finite element method is utilized to extract the Young’s modulus, the Poisson’s ratio, and the mass density values of each layer material of a bilayer tablet from a subset of the measured resonance frequencies. For verification purposes, a contact ultrasonic technique based on the time-of-flight data of the longitudinal (pressure) and transverse (shear) acoustic waves in each layer of a bilayer tablet is also utilized. The extracted mechanical properties from the air-coupled acoustic data agree well with those determined from the contact ultrasonic measurements. The mechanical properties of solid oral dosage forms have been shown to impact its mechanical integrity, disintegration profile and the release rate of the drug in the digestive tract, thus potentially affecting its therapeutic response. The presented nondestructive technique provides greater insight into the mechanical properties of the bilayer tablets and has the potential to identify quality and performance problems related to the mechanical properties of the bilayer tablets early on the production process and, consequently, reduce associated cost and material waste.     

Guidelines for the Fitting of Anomalous Diffusion Mean Square Displacement Graphs from Single Particle Tracking Experiments

Single particle tracking is an essential tool in the study of complex systems and biophysics and it is commonly analyzed by the time-averaged mean square displacement (MSD) of the diffusive trajectories. However, past work has shown that MSDs are susceptible to significant errors and biases, preventing the comparison and assessment of experimental studies. Here, we attempt to extract practical guidelines for the estimation of anomalous time averaged MSDs through the simulation of multiple scenarios with fractional Brownian motion as a representative of a large class of fractional ergodic processes. We extract the precision and accuracy of the fitted MSD for various anomalous exponents and measurement errors with respect to measurement length and maximum time lags. Based on the calculated precision maps, we present guidelines to improve accuracy in single particle studies. Importantly, we find that in some experimental conditions, the time averaged MSD should not be used as an estimator.     

In Vitro–In Vivo Correlation of Efavirenz Tablets Using GastroPlus®

The aim of the present work was to use GastroPlus™ software for the prediction of pharmacokinetic profiles and in vitro–in vivo correlation (IVIVC) as tools to optimize the development of new generic medications. GastroPlus™ was used to simulate the gastrointestinal compartment and was based on the advanced compartmental absorption and transit model. Powder dissolution and efavirenz tablet dissolution studies were carried out to generate data from which correlation was established. The simulated plasma profile, based on the physicochemical properties of efavirenz, was almost identical to that observed in vivo for biobatches A and B. A level A IVIVC was established for the dissolution method obtained for the generic candidate using the Wagner–Nelson (r² = 0.85) and for Loo–Riegelman models (r² = 0.92). The percentage of fraction absorbed indicated that 0.5% sodium lauryl sulfate may be considered a biorelevant dissolution medium for efavirenz tablets. The simulation of gastrointestinal bioavailability and IVIVC obtained from immediate-release tablet formulations suggests that GastroPlus™ is a valuable in silico method for IVIVC and for studies directed at developing formulations of class II drugs.KEY WORDS: bioavailability, computational simulation, efavirenz, GastroPlus™, in vivo–in vitro correlation     

Hydrophilic excipients modulate the time lag of time-controlled disintegrating press-coated tablets

An oral press-coated tablet was developed by means of direct compression to achieve the time-controlled disintegrating or rupturing function with a distinct predetermined lag time. This press-coated tablet containing sodium diclofenac in the inner core was formulated with an outer shell by different weight ratios of hydrophobic polymer of micronized ethylcellulose (EC) powder and hydrophilic excipients such as spray-dried lactose (SDL) or hydroxypropyl methylcellulose (HPMC). The effect of the formulation of an outer shell comprising both hydrophobic polymer and hydrophilic excipients on the time lag of drug release was investigated. The release profile of the press-coated tablet exhibited a time period without drug release (time lag) followed by a rapid and complete release phase, in which the outer shell ruptured or broke into 2 halves. The lag phase was markedly dependent on the weight ratios of EC/SDL or EC/HPMC in the outer shell. Different time lags of the press-coated tablets from 1.0 to 16.3 hours could be modulated by changing the type and amount of the excipients. A semilogarithmic plot of the time lag of the tablet against the weight ratios of EC/SDL or EC/HPMC in the outer shell demonstrated a good linear relationship, withr=0.976 andr=0.982, respectively. The predetermined time lag prior to the drug release from a press-coated tablet prepared by using a micronized EC as a retarding coating shell can be adequately scheduled with the addition of hydrophilic excipients according to the time or site requirements.     

Epidemiology of musculoskeletal disorders among computer users: lesson learned from the role of posture and keyboard use.

Reports in the scientific literature and lay press have suggested that computer users are at increased risk of upper extremity musculoskeletal disorders (MSDs). Early studies often found elevated rates of MSD outcomes among keyboard users when compared to non-users. Attention soon focused on specific aspects of keyboard work that might be responsible for the observed rate increase. In this review, the epidemiological evidence examining associations between MSD outcomes and computer user posture and keyboard use intensity (hours of computer use per day or per week) are examined. Results of epidemiological studies of posture and MSD outcomes have not been entirely consistent. Reasons for the inconsistency in results include cross-sectional study design (with possible failure to assure that measured exposure preceded health effect), imprecision of posture measures used, and difficulties involved in analyzing multiple related variables. Despite the inconsistencies, it appears from the literature that posture is an independent risk factor of modest magnitude for MSDs among computer users. It appears that lowering the height of the keyboard to or below the height of the elbow and resting the arms on the desk surface or chair armrests is associated with reduced risk of neck and shoulder MSDs. Results of epidemiological studies examining computer use (hours keying per day or per week) are more consistent than those examining posture, although some inconsistency is observed. Reasons for the inconsistency include possible selective survival bias resulting from cross-sectional study design, differences in exposure categorization, and possible interaction with other exposure variables. Overall, the literature shows that daily or weekly hours of computer use is more consistently associated with hand and arm MSDs than with neck and shoulder MSDs.     

Assessment of NIR spectroscopy for nondestructive analysis of physical and chemical attributes of sulfamethazine bolus dosage forms

The goal of this study was to assess the utility of near infrared (NIR) spectroscopy for the determination of content uniformity, tablet crushing strength (tablet hardness), and dissolution rate in sulfamethazine veterinary bolus dosage forms. A formulation containing sulfamethazine, corn starch, and magnesium stearate was employed. The formulations were wet granulated with a 10% (wt/vol) starch paste in a high shear granulator and dried at 60°C in a convection tray dryer. The tablets were compressed on a Stokes B2 rotary tablet press running at 30 rpm. Each sample was scanned in reflectance mode in the wavelengths of the NIR region. Principal component analysis (PCA) of the NIR tablet spectra and the neat raw materials indicated that the scores of the first 2 principal components were highly correlated with the chemical and physical attributes. Based on the PCA model, the significant wavelengths for sulfamethazine are 1514, (1660–1694), 2000, 2050, 2150, 2175, 2225, and 2275 nm; for corn starch are 1974, 2100, and 2325 nm; and for magnesium stearate are 2325 and 2375 nm. In addition, the loadings show large negative peaks around the water band regions (≈1420 and 1940 nm), indicating that the partial least squares (PLS) models could be affected by product water content. A simple linear regression model was able to predict content uniformity with a correlation coefficient of 0.986 at 1656 nm; the use of a PLS regression model, with 3 factors, had anr ² of 0.9496 and a sandard error of calibration of 0.0316. The PLS validation set had anr ² of 0.9662 and a standard error of 0.0354. PLS calibration models, based on tablet absorbance data, could successfully predict tablet crushing strength and dissolution in spite of varying active pharmaceutical ingredient (API) levels. Prediction plots based on these PLS models yielded correlation coefficients of 0.84 and 0.92 on independent validation sets for crushing strength and Q₁₂₀ (percentage dissolved in 120 minutes), respectively. Published: September 20, 2005 The opinions expressed in this paper are of the authors' personal views. They do not necessarily reflect the views or policies of the FDA.     

Motion characterization by self-distribution–function procedure

In the present paper a procedure for the biomolecular motion characterization based on the evaluation of the Mean Square Displacement (MSD), through the Self Distribution Function (SDF), is presented. In particular it will be shown how the MSD, which represents a good observable for the characterization of the dynamical properties in disordered systems, can be decomposed into partial contributions associated to the system dynamical processes within a specific spatial scale. It will be shown how the SDF procedure allows to evaluate both the total MSD and the partial MSDs through the total SFD and the partial SDFs. As a result, the total MSD is the weighed sum of the partial MSD contributions in which the weights are obtained by the fitting procedure of measured EINS intensity data. We apply the SDF procedure at EINS data collected, by the IN13 backscattering spectrometer at the Institute Laue-Langevin, Grenoble, on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry myoglobin in trehalose environment. It emerges that the hydrogen bond imposed network of the water–trehalose mixture appears to be stronger with respect to that of the water–sucrose mixture and this result can justify the highest bioprotectant effectiveness of trehalose in comparison with sucrose. Furthermore it emerges that, the partial MSD behaviours of sucrose and trehalose are equivalent in the low Q domain (0–1.7) Å^− 1 whereas they are different in the high Q domain (1.7–4) Å^- 1. This circumstance suggests that the higher structure sensitivity of sucrose in respect to trehalose should be related to the small spatial observation windows.     

基于实地调查和高光谱数据的浑善达克沙地中部植物alpha多样性遥感估测

植物群落物种多样性的快速无损估测一直是近几十年生态学领域的热点研究问题。相对于大尺度的卫星遥感数据,高光谱遥感数据具有光谱和空间分辨率高的优势。采用ASD HH2便携式高光谱仪,收集浑善达克沙地中部120个样方的高光谱数据,并对样方的alpha多样性指数进行同步测定。对高光谱遥感数据进行预处理,采用相关性分析、主成分分析和经验波段筛选法,从数百个波段中选择敏感波段。采用90个样方的高光谱数据作为训练样本,对筛选的敏感波段进行多元线性逐步回归分析,获得12个回归模型。采用另外30个样方的高光谱数据作为验证样本,对回归模型的拟合效果进行检验。结果发现,采用主成分分析法提取敏感波段的回归模型拟合效果最好,Pielou指数、Shannon-Wiener指数和Simpson指数拟合均达到显著水平。对我国植物物种多样性微尺度的快速评估和高光谱遥感具有一定参考意义,并对未来植物多样性高光谱遥感研究提出了建议。     

Evaluation of the Performance Characteristics of Bilayer Tablets: Part II. Impact of Environmental Conditions on the Strength of Bilayer Tablets

Ambient air humidity and temperature are known to influence the mechanical strength of tablets. The objective of this work is to understand the influence of processing parameters and environmental conditions (humidity and temperature) on the strength of bilayer tablets. As part of this study, bilayer tablets were compressed with different layer ratios, dwell times, layer sequences, material properties (plastic and brittle), first and second layer forces, and lubricant concentrations. Compressed tablets were stored in stability chambers controlled at predetermined conditions (40C/45%RH, 40C/75%RH) for 1, 3, and 5 days. The axial strength of the stored tablets was measured and a statistical model was developed to determine the effects of the aforementioned factors on the strength of bilayer tablets. As part of this endeavor, a full 3 × 2⁴ factorial design was executed. Responses of the experiments were analyzed using PROC GLM of SAS (SAS Institute Inc, Cary, North Carolina, USA). A model was fit using all the responses to determine the significant interactions (p < 0.05). Results of this study indicated that storage conditions and storage time have significant impact on the strength of bilayer tablets. For Avicel–lactose and lactose–Avicel tablets, tablet strength decreased with the increasing humidity and storage time. But for lactose–lactose tablets, due to the formation of solid bridges upon storage, an increase in tablet strength was observed. Significant interactions were observed between processing parameters and storage conditions on the strength of bilayer tablets.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-012-9846-8) contains supplementary material, which is available to authorized users.KEY WORDS: axial tester, bilayer tablet, design of experiments, storage conditions, tablet strength     

Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl⁻ channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRΔF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.     

Gender differences in neck/shoulder muscular patterns in response to repetitive motion induced fatigue

Previous studies have associated amplitude and frequency characteristics of the electromyogram (EMG) to the risk of developing musculoskeletal disorders (MSDs) with repetitive tasks. However, few studies have investigated whether EMG variability and between-muscle activity characteristics may be associated with MSD risk. Twenty-six healthy volunteers (13 men, 13 women) performed a repetitive pointing task at shoulder height until scoring 8 on a Borg CR-10 scale. Electromyographic (EMG) signals were recorded from six neck/shoulder muscle sites. EMG amplitude (RMS), variability and mutual information (MI) among muscle pairs were computed. Muscle fatigue was evidenced by increased EMG RMS of four muscles (Upper Trapezius (UT): +17%; supraspinatus (SUPRA): +28%; middle deltoid: +13%; biceps brachii: +38%) and increased SUPRA variability. Correlations between minute 1 patterns and endurance time indicated that in women, initially high variability in UTR (r = 0.79) and SUPRA (r = 0.71) predicted higher endurance, whereas in men, initially low MI in LT–UT (?0.69) and in LT–SUPRA (?0.77) pairs predicted high endurance. Significant correlations suggest that variability and between-muscle patterns may be associated with fatigue and injury mechanisms, in a gender-specific way. Differing fatigue mechanisms between genders could help explain gender differences in injury mechanisms.     

Transformation of Mucor circinelloides with Autoreplicative Vectors Containing Homologous and Heterologous ARS Elements and the Dominant Cbx r Carboxine-Resistance Gene

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu⁺ Cbx^r) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx^r(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 μ plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65–120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.     

Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: Total and free testosterone reference ranges

Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low-testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15 min per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150 μL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3 ng/dL, and the assay was linear up to 2000 ng/dL. The method compared very well with an established RIA: y = 1.02x + 1.5, r² = 0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pediatric reference ranges were developed for this very sensitive and accurate high-throughput LC-MS/MS method. This method is suitable for measuring the expected low-testosterone concentrations seen in women, children, and hypogonadal males and for monitoring testosterone suppressive therapy in prostate cancer patients.