PCR-free digital minisatellite tandem repeat genotyping

We demonstrated a proof‐of‐concept for novel minisatellite tandem repeat typing, called PCR‐free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a ligation reaction instead of PCR thermal cycling, magnetic bead‐based solid‐phase capture for purification, and an elongated sample stacking microcapillary electrophoresis (μCE) for sensitive digital coding of repeat number. We designed a 16‐bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin‐coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min.     

Elimination of the uninformative calibration sample subset in the modified UVE(Uninformative Variable Elimination)-PLS (Partial Least Squares) method.

In order to increase the predictive ability of the PLS (Partial Least Squares) model, we have developed a new algorithm, by which uninformative samples which cannot contribute to the model very much are eliminated from a calibration data set. In the proposed algorithm, uninformative wavelength (or independent) variables are eliminated at the first stage by using the modified UVE (Uninformative Variable Elimination)-PLS method that we reported previously. Then, if the prediction error of the ith (1 < or =i< or = n) sample is larger than 3sigma, the corresponding sample is eliminated as uninformative, where n is the total number of calibration samples and sigma is the standard deviation calculated from the other n(-1) samples. Calculation of sigma by the leave-one-out manner enhances the ability to identify the uninformative samples. The final PLS model is constructed precisely because both uninformative wavelength variables and uninformative samples are eliminated. In order to demonstrate the usefulness of the algorithm, we have applied it to two kinds of mid-infrared spectral data sets.     

An inter‐laboratory cross‐validation study for the determination of perampanel in human plasma by liquid chromatography assays

For sample assay to support global clinical studies of perampanel, a novel AMPA receptor antagonist, six chromatographic assay methods in human plasma were developed and fully validated at each laboratory using liquid chromatography with tandem mass spectrometry (LC‐MS/MS) or LC with fluorescence detection (LC‐FL). In this study, samples fortified with known perampanel concentrations were assayed at six laboratories to find whether assay data are comparable. Perampanel was extracted by protein precipitation or liquid–liquid extraction, chromatographed on a reverse‐phase column then detected by MS/MS or FL to achieve the limit of quantification of 0.25 or 1 ng/mL. Cross‐validation samples at four concentrations prepared at a central laboratory were determined at six laboratories and the mean accuracy at each concentration was within ±15% except the low concentration at one laboratory (relative error ?17.4%), suggesting that plasma concentrations of perampanel in clinical trials can be compared across laboratories.     

Strategies for constructing the calibration set for a near infrared spectroscopic quantitation method

Three strategies for the construction of calibration sets have been tried, with the objective to develop and to validate a NIR quantitation method.The first two approaches consist of the use of two types of samples, named: samples of laboratory obtained by mixing the ingredients that compose the drug, and doped samples obtained by under- and over-dosed production samples. In order to improve the prediction results, production samples have been added to each calibration model. The ensuing models were validated with a view to determine their fitness for purpose. However, spectral differences between the laboratory samples and doped samples resulted in spurious predictions in quantifying samples of one type using the model developed from samples of the other.Such differences were studied in depth and a third procedure has been proposed, based on a calibration model constructed with an unique type of sample (laboratory sample) for later to correct it with a few doped samples. This corrected model has a good predictive ability on production samples.     

Incurred sample reanalysis in drug discovery bioanalysis

Bioanalysis plays a key role during the drug discovery process to generate the pharmacokinetic data to facilitate unbiased evaluation of leads, optimized leads and drug candidates. Such pharmacokinetic data are used to enable key decisions in the drug discovery process. The aim of the work is to put forward a new strategy of performing the incurred sample reanalysis for select small molecule novel chemical entities at different stages of drug discovery prior to candidate selection. Three discovery programs representing hits, leads and optimized lead candidates were selected for the incurred sample reanalysis (ISR) analysis. From each discovery program, two novel chemical entities were selected for the ISR analysis. The time points considered for ISR generally varied among the programs; however, samples coinciding with drug absorption, distribution and elimination were considered in the ISR assessment. With the exception of a single ISR value that gave a high deviation (about 63%), the observed ISR values supported the discovery bioanalytical assays. While the individual bioanalytical laboratory can draw an algorithm for selecting novel chemical entities and fixing the acceptance criteria for the ISR data, it is proposed that the percentage difference between ISR vs. original concentration for 67% of the repeat samples is contained within ±30% for discovery bioanalysis.     

Microdevice DNA forensics by the simple tandem repeat method

We review recent experiments on DNA forensics by the simple tandem repeat (STR) method using a 16-lane micromachined device as the active separation element. Separations by linear polyacrylamide matrices show very high data quality metrics when evaluated with statistically significant data sets. Full 16-locus multiplexes are verified on the multilane system. Multi-donor mixed samples are studied in the context of the limits of the laser-induced fluorescence detector and data-reduction software. The microdevice appears to be posed to outperform current capillary arrays in terms of stability and, through specialized sample loading, in the interpretation of complex mixtures.     

Locus-specific brackets for reliable typing of Y-chromosome short tandem repeat markers

Short tandem repeat (STR) loci, widely used as genetic markers in disease diagnostic studies and human identity applications, are traditionally genotyped through comparison of allele sizes to a sequenced allelic ladder. Allelic ladders permit a floating bin allele calling method to be utilized, which enables reliable allele calling across laboratories, instrument platforms, and electrophoretic conditions. Precise sizing methods for STR allele calling involving fixed bins can also be used when a high degree of precision has been demonstrated within an instrument platform and a set of electrophoretic conditions. An alternative method for reliable genotyping of STR markers, locus-specific brackets (LSBs), is introduced here. LSBs are artificial alleles created through molecular biology manipulations to be shorter or longer than alleles commonly seen in populations under investigation. The size and repeat number of measured alleles are interpolated between the two LSB products that are mixed with the polymerase chain reaction-amplified STR alleles. The advantages and limitations of the LSB approach are described along with a concordance study between the LSB typing approach and other STR typing methods. Complete agreement was observed with 162 samples studied at 5 Y-chromosome loci.     

Digital simulation as an aid to sample scheduling in a routine laboratory for liquid chromatography

A computer model is described to aid decision-making in a routine laboratory for liquid chromatography. The managerial demand was to reduce the delay times of the samples in the laboratory despite an increasing sample input. Knowledge derived from records on the laboratory and from the experience of the laboratory staff is used to develop a simulation model of the laboratory. The model is applied to study the effects of several factors on the performance of the laboratory in order to trace the bottlenecks.     

Analysis of multiple pesticide residues in tobacco using pressurized liquid extraction, automated solid-phase extraction clean-up and gas chromatography-tandem mass spectrometry

A new method was developed for the analysis of pesticide residues in tobacco. The objective was to significantly increase the number of samples that can be processed by the laboratory and to enable the extension of the current coverage to additional pesticides. A new analytical approach was therefore defined based on two main axes, the automation of the sample preparation and the selectivity of the analyte detection using tandem mass spectrometry. This latter aspect reduces the stringency of the requirements placed on the clean-up of the extracts and on the chromatographic resolution when less selective detectors are used. The extraction of the analytes from the matrix is performed using the pressurized liquid extraction technique. Tobacco samples are extracted at elevated temperature and pressure (100 C and 100 atm; 1 atm = 101,325 Pa) using acetone as an extraction solvent. The resulting extract is then concentrated using a Vortex evaporator. Three different solid-phase extraction (SPE) procedures, adjusted to the chemical properties of the different active ingredients to be measured, are applied to the concentrated extract, thus leading to three extract fractions. The first fraction contains such main classes of active ingredients as organohalogenated and 2,6-dinitroaniline compounds while the second one collects the organophosphorus and acylalanines residues; these two fractions are analyzed by capillary gas chromatography coupled to tandem mass spectrometry using negative chemical ionization and electron impact ionization in the positive mode, respectively. The third extract fraction gathers the N-methylcarbamates residues which are analyzed by HPLC with post-column derivatization and fluorescence detection. The different sample preparation stages from extraction to SPE clean-up have been automated through the use of recent analytical technologies. In combination with the analysis by tandem mass spectrometry, this provided a potential for a high sample throughput.     

Validation of the HumDXS6807 short tandem repeat polymorphism for forensic application.

This paper presents sequence and population genetic data of the HumDXS6807 short tandem repeat (also known as CHLC.GATA52B03) which is a tetranucleotide repeat polymorphism representing seven alleles of 251-275 bp in length. HumDXS6807 is located at Xpter-Xp22.2, i.e., at a genetic distance of more than 87 and 151 cM from the well-known markers HumARA and HumHPRTB, respectively. Kinship tests in 157 family trios revealed a typical X-linked codominant inheritance; mutations were not found. Population genetic data were obtained by analyzing a Caucasian population sample comprising 308 females and 209 males: polymorphism information content (PIC) = 0.640; Heterozygosity (Het) = 0.668; Mean exclusion chance (MEC) = 0.414. The HumDXS6807 allele distribution met the Hardy-Weinberg expectations.     

Post-column infusion study of the 'dosing vehicle effect' in the liquid chromatography/tandem mass spectrometric analysis of discovery pharmacokinetic samples

It has become increasingly popular in drug development to conduct discovery pharmacokinetic (PK) studies in order to evaluate important PK parameters of new chemical entities (NCEs) early in the discovery process. In these studies, dosing vehicles are typically employed in high concentrations to dissolve the test compounds in dose formulations. This can pose significant problems for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of incurred samples due to potential signal suppression of the analytes caused by the vehicles. In this paper, model test compounds in rat plasma were analyzed using a generic fast gradient LC/MS/MS method. Commonly used dosing vehicles, including poly(ethylene glycol) 400 (PEG 400), polysorbate 80 (Tween 80), hydroxypropyl beta-cyclodextrin, and N,N-dimethylacetamide, were fortified into rat plasma at 5 mg/mL before extraction. Their effects on the sample analysis results were evaluated by the method of post-column infusion. Results thus obtained indicated that polymeric vehicles such as PEG 400 and Tween 80 caused significant suppression (> 50%, compared with results obtained from plasma samples free from vehicles) to certain analytes, when minimum sample cleanup was used and the analytes happened to co-elute with the vehicles. Effective means to minimize this 'dosing vehicle effect' included better chromatographic separations, better sample cleanup, and alternative ionization methods. Finally, a real-world example is given to illustrate the suppression problem posed by high levels of PEG 400 in sample analysis, and to discuss steps taken in overcoming the problem. A simple but effective means of identifying a 'dosing vehicle effect' is also proposed.     

Characterization of UVC-induced DNA damage in bloodstains: forensic implications

The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA–DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.     

RDF法对非晶态聚醚砜共聚物的近程有序结构研究

用径向分布函数(RDF)研究了聚醚砜共聚物(PES)在r=1.5 nm范围内的近程结构, 结果表明, 分子链间存在近程有序结构, 有序周期约为0.5 nm; 聚醚砜共聚物经退火处理后, 由g(r)图可以看出, 第一个峰变高、变窄; 由G(r)图可以看出, 分子链间第一个有序区内, 分子链间贡献大于链内贡献; 于200 ℃退火处理后有序畴尺寸(rs)略有增大, 原子平均位移(σ)变小, 使得分子链内原子间距分布更加均匀, 相邻分子链间距(rv)变小. 同时透射电镜(TEM)的形态结构分析表明, 经退火处理后, 其聚集态确实形成了某种局部有序的结构.     

Uncertainty calculations in the certification of reference materials. 2. Homogeneity study

 Many reference materials undergo a batch certification, which implies that a small number of samples is taken from a batch, characterised, and these results are then assumed to be representative of all remaining samples. An important aspect in this design is the translation of the characterisation data to a single sample, as usually the laboratory will be using only one sample of the batch. This form of homogeneity is very important and can be influenced to a certain extent by well-designed sample preparation procedures. Another subsampling problem associated with many reference materials is that only a small test portion is drawn from the sample to carry out the measurement. Obviously, this test portion must be representative of the sample, otherwise the certified value is still not applicable. Both kinds of homogeneity tests are examined in the paper and evaluated using practical examples. Received: 31 May 2000 · Accepted: 29 July 2000     

Determination of drug concentrations in plasma by a highly automated, generic and flexible protein precipitation and liquid chromatography/tandem mass spectrometry method applicable to the drug discovery environment

This report presents a highly automated procedure for the determination of drug concentrations in plasma samples. The method is generic, in that it has been applied without adaptation to many different drug candidate molecules, but is also flexible, in that variations in the nature and number of samples to be analyzed can be readily accommodated. The method includes preparation of dilutions of analyte stock solutions, spiking these into control plasma to generate analytical standards, and preparation of samples suitable for analysis by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) by precipitation of plasma proteins with acetonitrile, centrifugation, and dilution of the supernatants with HPLC buffer. All of these steps, apart from centrifugation, are performed without manual intervention on an automated liquid-handling workstation using 96-well plates. Analysis is by HPLC/MS/MS, using a generic HPLC gradient. Commercially available software was used for optimization of parameters for analysis by HPLC/MS/MS, integration of chromatographic peaks, and quantification of drug concentrations. The use of this methodology in our laboratory has greatly facilitated the analysis of small sample sets for a large number of analytes, a situation regularly encountered in an early drug discovery environment.     

Precision and accuracy in fluorescent short tandem repeat DNA typing: assessment of benefits imparted by the use of allelic ladders with the AmpF/STR Profiler Plus kit

Base-calling precision of short tandem repeat (STR) allelic bands on dynamic slab-gel electrophoresis systems was evaluated. Data was collected from over 6000 population database allele peaks generated from 468 population database samples amplified with the AmpF/STR Profiler Plus (PP) kit and electrophoresed on ABD 377 DNA sequencers. Precision was measured by way of standard deviations and was shown to be essentially the same, whether using fixed or floating bin genotyping. However, the allelic ladders have proven more sensitive to electrophoretic variations than database samples, which have caused some floating bins of D18S51 to shift on occasion. This observation prompted the investigation of polyacrylamide gel formulations in order to stabilize allelic ladder migration. The results demonstrate that, although alleles comprised in allelic ladders and questioned samples run on the same gel should migrate in an identical manner, this premise needs to be verified for any given electrophoresis platform and gel formulation. We show that the compilation of base-calling data is a very informative and useful tool for assessing the performance stability of dynamic gel electrophoresis systems, stability on which depends genotyping result quality.     

Optimizing an ultrafast generic high-performance liquid chromatography/tandem mass spectrometry method for faster discovery pharmacokinetic sample throughput

For higher throughput screening, where the number of new chemical entities (NCEs) to test is rapidly increasing, fast sample turnaround time is essential. In order to increase efficiency a generic high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method, with a cycle time of 85 s (42 injections/h), was created. This was accomplished through the use of a 1-min ballistic gradient and the optimization of the autosampler. The gradient was optimized by varying the organic mobile phase concentration and examining its ballistic characteristics with respect to matrix ion suppression and compound retention time. The autosampler time could be reduced by optimizing several parameters and by determining the source of most of the carryover in order to reduce the number of syringe and injector washes. Finally, the reliability of the new generic method is demonstrated by comparison of sample data with a standard 2-min linear gradient method that showed that the data sets were well correlated. For plasma AUC (ng.h/mL) of 28 NCEs, the regression line had a slope of 0.92 and the R2 was 0.929. The described method was found to be useful for both rat plasma and tissue samples.     

Chemometric approach to open validation protocols: Prediction of validation parameters in multi-residue ultra-high performance liquid chromatography–tandem mass spectrometry methods

The recent technological advancements of liquid chromatography–tandem mass spectrometry allow the simultaneous determination of tens, or even hundreds, of target analytes. In such cases, the traditional approach to quantitative method validation presents three major drawbacks: (i) it is extremely laborious, repetitive and rigid; (ii) it does not allow to introduce new target analytes without starting the validation from its very beginning and (iii) it is performed on spiked blank matrices, whose very nature is significantly modified by the addition of a large number of spiking substances, especially at high concentration. In the present study, several predictive chemometric models were developed from closed sets of analytes in order to estimate validation parameters on molecules of the same class, but not included in the original training set. Retention time, matrix effect, recovery, detection and quantification limits were predicted with partial least squares regression method. In particular, iterative stepwise elimination, iterative predictors weighting and genetic algorithms approaches were utilized and compared to achieve effective variables selection. These procedures were applied to data reported in our previously validated ultra-high performance liquid chromatography–tandem mass spectrometry multi-residue method for the determination of pharmaceutical and illicit drugs in oral fluid samples in accordance with national and international guidelines. Then, the partial least squares model was successfully tested on naloxone and lormetazepam, in order to introduce these new compounds in the oral fluid validated method, which adopts reverse-phase chromatography. Retention time, matrix effect, recovery, limit of detection and limit of quantification parameters for naloxone and lormetazepam were predicted by the model and then positively compared with their corresponding experimental values. The whole study represents a proof-of-concept of chemometrics potential to reduce the routine workload during multi-residue methods validation and suggests a rational alternative to ever-expanding procedures progressively drifting apart from real sample analysis.     

Strategies in molecular spectroscopic analysis with application of queueing theory and digital simulation

Waiting lines in a laboratory for molecular spectroscopie analysis are simulated by digital methods. The influence of several parameters, such as the mean analysis time, the mean inter-arrival time of the samples and the routing procedure are evaluated by application of queueing theory. The queues were treated as a sample realization from a stationary first-order autoregressive system. The structure of the time series of the number of waiting samples in the model was identical to the time series observed in a real laboratory. The possibilities of digital simulation for decision-making in analytical laboratories.are demonstrated.