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Data from 166 patients who had completed 6 months of anakinra (Kineret, r-metHuIL-1Ra) therapy under the conditions of daily clinical practice showed an efficacy and safety profile comparable to the data known from clinical trials. Patients significantly responded as early as 1 month following initiation of therapy. The data suggest that anakinra may also be effective in patients who have failed tumor necrosis factor (TNF)-blocking agents. Injection-site reactions were reported less frequently than in clinical trials, indicating that these reactions seem to require less therapeutic attention under non-clinical trial conditions.

Out of a total of 624 consecutive gunshot autopsies from Münster and Hamburg, Germany, 32 cases (5.1%) were accidental. The accidents were self-inflicted in 3 cases while another person fired the gun in the remaining 29 cases. More than half of the victims were younger than 25 years and 75% were male. A single gunshot injury was present in all cases and the head was struck in 47% but a detailed analysis of the entrance wound sites did not show any preferential anatomical sites. A surprising finding was the presence of five contact or near contact gunshots (16%). The reasons for these and most other accidents were extreme carelessness when handling a firearm, the involvement of children or adolescents or a foolish behaviour with a gun intended to impress others. Gun-cleaning accidents occurred rarely and there were no major technical defects of the weapons. Preventive measures should concentrate on strict inaccessibility of guns to children and on increased educational efforts to subgroups at risk such as hunters and members of the armed forces. A single non-contact gunshot injury from a long-barrelled firearm can be considered typical for an accident but the great variety and the possible presence of "disguised" suicides and homicides requires a careful forensic investigation including inspection of the scene and reconstruction of the events. It is recommended that a case should always be considered to be non-accidental in the beginning of an investigation.

A case of malignant hyperthermia (mh) in a 27-year-old man is described. In a first anaesthesia using isoflurane and succinylcholine, the end-tidal CO(2) rose from 39 to 49 mmHg 2.75 h post-intubation and the body temperature rose to 39.8 degrees C 14 h post-intubation but was normal again the next day. In a second anaesthesia using the same medication, the maximal end-tidal CO(2) was 44 mmHg and the body temperature rose to 39 degrees C after 9 h. After 4 days, the fever rose to 40 degrees C, and to 42 degrees C when death occurred 10 days after the second anaesthesia. Masseter spasms or muscle rigidity were never present. According to the death certificate, death was due to multi-organ failure from sepsis. At autopsy, the skeletal muscles were pale and oedematous. Histology demonstrated focal necroses in the skeletal muscles, shock kidneys with myoglobin excretion and myoglobin clots in small blood vessels of the lungs. Hence, the postmortem diagnosis "malignant hyperthermia" was established but accusations of medical maltreatment were rejected because of the atypical and protracted clinical course and because uncharacteristic signs of malignant hyperthermia were attributable to the clinically suspected sepsis., (Copyright 2002 Elsevier Science Ireland Ltd.)

A total of 624 consecutive gunshot autopsies from the Institutes of Legal Medicine in Münster and Hamburg was investigated retrospectively. In a subsample of 284 suicides and 293 homicides (n=577), a large variety of features such as firearm, ammunition, number and site of entrance wounds, shooting distance and direction of the internal bullet path were recorded and binary logistic regression analysis performed in the case of bullet paths. Females constituted 26.3% of the homicide victims and 10.6% of the suicides. Short-barrelled firearms outnumbered long arms in homicides by 6:1 and in suicides by 2:1. More than 1 gunshot injury was found in 5.6% of the suicides (maximum 5 gunshots) and in 53.9% of the homicides (maximum 23 gunshots). The suicidal gunshots were fired from contact or near contact range in 89% while this was the case in only 7.5% of the homicides. The typical entrance wound sites in suicides were the temple (36%), mouth (20%), forehead (11%) and left chest (15%) but uncommon entrance wound sites such as the eye, ear, and back of the neck and head were also encountered. In suicidal gunshots to the right temple (n=107), only 6% of the bullet paths were directed downwards and only 4% were directed from back-to-front. In gunshots to the left chest (n=130), bullet paths running right-to-left or parallel occurred frequently in suicides (75%) and infrequently in homicide victims (19%). From 61 suicides who fired the gun inside their mouth, only 1 pointed the gun downwards. Consequently, some bullet path directions cannot be considered indicative of suicide: downwards and back-to-front in gunshots to the temple, left-to-right in gunshots to the left chest and downwards in mouth shots. The isolated autopsy findings can only be indicative of suicide or homicide but the combined analysis of several findings can be associated with a high probability.

The deposition of backspatter on the firearm or person shooting can greatly assist the reconstruction of shooting incidents. Backspatter was investigated in experimental transverse gunshots (9-mm Luger) to the heads of calves (n = 9) from shooting distances of 0 to 10 cm. The firearms were examined with a magnifying glass; the surgical gloves and the right sleeve worn by the person shooting were examined with a stereomicroscope. On the firearms, backspatter of blood was found in five of the nine cases, and one or both gloves showed bloodspatter deposits in six and the right sleeves in four cases. Most droplets were 1 to 3 mm and circular or elongated. In addition, a fine spray of tiny blood deposits was present on the firearm and textiles in four cases. The distribution of the droplets on the firearms varied: the areas included regions shielded by prominent parts, and the droplets were predominantly located on the extensor side of the fingers and the radial aspect of the hands and sleeves. Backspatter of tiny bone fragments was recovered from the firearm and sleeve in only one case, but tissue (bone, fat, muscle, skin) was present on the ground in front of the entrance wound in seven cases. A careful investigation, including appropriate lightning and magnification, is necessary for reliable statements concerning the absence of backspatter or the extent of backspatter present.

Fatalities caused by electrocution (n = 37) were re-examined on the basis of the autopsy records, the police reports and the technical expertise. Accidents including two lightning deaths caused 2/3rd and suicides 1/4th of the fatalities and there were two homicides. Carelessness or ignorance were a major reason for accidental deaths while technical defects could be verified in five cases only. Low-voltage ac and male victims predominated. Electrical burns or current marks were detected in 81% including all high voltage electrocutions. The seven victims lacking burns were electrocuted in a wet environment including six cases of suicide in the bathtub with a hair drier. Petechial haemorrhages were present in 74% of the cases and the favourite sites were the skin of the eyelids, conjunctivae, visceral pleura, and the epicard. The presence of petechiae did not depend on the voltage or the current pathway relative to the heart. It is, therefore, suggested that the petechiae are not caused by asphyxia but by a combination of venous congestion due to cardiac arrest and a sudden rise in blood pressure induced by muscle contractions. Consequently, "electrical petechiae" represent a non-specific but typical finding in electrocution irrespective of the mechanism leading to death. Unlike electrical burns, petechiae also indicate the vital origin of the events. The relevance of this typical morphological sign in the examination of possible electrocution fatalities is therefore emphasised., (Copyright 2002 Elsevier Science Ireland Ltd.)

Two fatalities due to unusual impalement injuries are reported. (1) A large branch broken off during a storm had entered a passing car and perforated the chest of the driver and the back of the seat. The chest organs were grossly lacerated. The car was subsequently stopped by another tree and this second impact removed the wood from the body. (2) A man suffered anorectal impalement by the leg of a stool turned upside down. He had introduced one stool leg into his anus for sexual stimulation and fell onto it. This resulted in a wound channel 36 cm long including perforation of the rectum, urinary bladder, mesentery, transverse mesocolon and liver. Before autopsy, the mode of death was unclear because the man had removed the stool leg himself, his wife had hidden the stool from the scene, and there were no relevant external injuries. In both cases, a reliable reconstruction required investigation of the scene and consideration of extremely unlikely circumstances or of bizarre human activities.

In 1945 a 6-year-old girl received a tangential gunshot injury to the left posterior inferior skull. After hospitalisation because of septicemia she was unable to walk for several months. Since 1967 she had been repeatedly applying for compensation because of pseudoneurasthenia and residual ataxia and many medical examinations were performed including X-ray, CT and MRI. Although certain objective findings (e. g. cerebellar atrophy) could be verified, a causal connection between the gunshot injury and the clinical findings could not be established. Therefore previous investigators concluded on a vascular origin of the disease. During the present re-examination of the patient, the authors found patch-like scars at the left posterior inferior skull base and behind the left ear, a cord-like scar formation in the subcutaneous tissue, connecting both skin scars, a gutter-like defect in the left occipital base of the skull and an indention of the left mastoid process, both again in line between the skin scars and a large defect of the left cerebellar hemisphere. It could be concluded with certainty that the anatomical findings and the clicinal symptoms were the direct result of a gunshot injury. Previous investigations had failed because of deficient investigations and techniques. In addition to an anatomical reconstruction, three dimensional reconstructions from CT scans proved most helpful.

In a retrospective evaluation of 799 consecutive autopsies of victims of sharp force performed between 1967 and 1996 in Münster and Berlin, only 18 cases (2.3%) were classified as accidents. A typical pattern was present in 15 cases: inebriated adults (1.4-3.6g/l BAC) fell into an architectural glass surface in the form of a door or window (12 cases), an aquarium, a mirrored wardrobe or a telephone cell. Another man fell into a large drinking glass. Many victims in this group showed multiple scratches, abrasions and superficial incisions as well as one or more deep tear/cut/puncture injury. The wound margins can be clean-cut or irregular and abraded. Death was mostly caused by exsanguination except for one case of air embolism and one case of cerebral injury. The fatal injuries were produced by large and dagger-like slivers of glass, by sharp-edged fragments of glass remaining inside the frame or by a portion of glass which fell down and acted in a way similar to a guillotine. Ordinary types of flat glass were involved in all cases and it is not until the impact that sharp fragments or cutting edges are produced. So the motion of the person commonly provides the force necessary for a fatal injury. This was also true for the remaining two cases not involving architectural glass. A farmer suffered cerebral injury from a fall into the long prong of a pitch fork, and the wounding agent was a knife in only one case. A man who stated that he had fallen into the knife in his hand died from pneumonia after inadequate therapy following a single stab injury to the periphery of the left lung and liver. Accidents where the victim is killed by his own knife therefore appear to be extremely rare.

A man committed suicide in his bathroom using a small pocket knife. At the autopsy a total of 92 stab wounds on the forehead, in both temples, the anterior aspect of the neck, the back of the neck, the chest and the sides of the trunk were found. In addition, repeated stabbing had caused a large soft tissue defect on the forehead. The frontal bone showed 3 perforations but no brain injury was present and two ribs were severed in the bony part, one of which carried a star-like pattern from repeated stabbing. No major vessels were injured and the cause of death was exsanguination after a considerable survival time. The large number of stab wounds, the perforation of bone and some injury sites, especially the head and back of the neck, are extraordinary findings in suicides which were probably favoured by insufficient anatomical knowledge and the use of a short-bladed knife. A psychiatric history could not be verified.

In cases of rape leading to fertilization, paternity testing can retrospectively identify the assailant. Abortion material commonly represents a mixture of maternal and fetal tissue and blood, which cannot be differentiated with the naked eye. Consequently, DNA typing of abortion material may be complicated, including band overlap if maternal tissue predominates. Therefore, histology screening of the abortion content for typical fetal tissue components, such as chorionic villi, followed by selected DNA typing of this sample is suggested. This combined approach is illustrated by a selected case demonstrating the reliability and concurrence of the histology and genetic results.

To illustrate how the high incidence of arrow wounds provided a major stimulus for the development of surgery until a century ago, we conducted a literature search. Our research shows that archaic peoples developed considerable surgical skill for extraction of arrows, including thoracotomy and trephination. A classical Hindu veda describes a variety of extraction methods, and Homer's Iliad introduces the term iatros, which means "he who extracts arrows." Hippocrates of Kos and Galen, representatives of the humoral doctrine, both shunned surgical intervention and considered purulence a drainage of materia peccans (spoiled humors). Cornelius Celsus was the first to systematically differentiate removal of arrows per extractionem and per expulsionem. Celsus recommended the spoon of Diocles, an ancient surgical instrument specially designed for extraction of arrows. Paulus of Aegina favored rapid extraction, aggressive therapy, and ligature on both sides of a vessel before extraction efforts. Paulus was the first to describe a special instrument for the removal of detached arrowheads per expulsionem (propulsorium). In medieval Europe, the standard of surgery declined drastically. The classical procedure under the dominant influence of the humoral concept was to await pus before extraction and to burn the wound with boiling oil and a branding iron. Arab authors had conserved the knowledge of Celsus and Paulus, and in Europe a renewal was achieved by Ambroise Paré, who has been called the creator of modern surgery. The incidence of arrow wounds increased once more in the American West. Joseph H. Bill, a famous U.S. Army Surgeon preoccupied with arrow wounds, favored rapid extraction and aggressive therapy, and he taught recruits not to apply traction on the shaft. The principles established by Celsus, Paulus, Paré, and Bill not only mark important landmarks in the evolution of surgery but can also serve as the basis for modern treatment of arrow wounds, which still occur, although on a small scale.

Three cases are presented where fatal puncture wounds caused by broken glass were very similar to stab wounds inflicted by a knife with a single-edged blade. Thus, all three cases caused a murder investigation to be initiated. It could only be determined that these wounds had been caused by glass after a detailed forensic autopsy. In two of the three cases, the only evidence for this was the identification of glass fragments in the wounds. The importance of X-ray examinations is underlined because modern glass in common use is radiopaque. Glass fragments lodged in the wounds can reduce the loss of blood and thus, prolong the capacity to act despite severe injuries.

We present the case of an 80-year-old malnourished and non-alcoholic woman who died from neglect-associated Marchiafava-Bignami disease, an illness usually almost exclusively occurring in male alcoholics. The patient had been bedridden for several months and had been looked after by her son. The patient was admitted to hospital in an extremely poor care condition suffering from severe exsiccosis, pressure sores and marasmus and died shortly afterwards. The initial post-mortem examination could not establish a definite cause of death, however, upon neuropathological examination a necrotising cystic lesion in the left cingulate gyrus as well as a central necrosis in the corpus callosum indicative of Marchiafava-Bignami disease were revealed. This is the first known case of Marchiafava-Bignami disease in a non-alcoholic woman and the first case in the forensic setting of neglect.

A .375 Holland & Holland Magnum Winchester Fail Safe bullet was fired against a steel plate. The big-game hunting bullet (17.4 g, approx. 810 m/s) has a solid copper-alloy hollow point design including a lead core limited to the rear portion. The range of firing was 20 m and the angle of impact was 90 degrees. A large fragment (10.9 g) consisting of the main part of the copper portion was deformed in a peculiar manner similar to a tube or ring and wounded the left hand of the person shooting. The unique fragment trajectory strictly against the line of fire and the velocity sufficient to shatter bone after a distance of 20 m can be explained by an extraordinary deformation mechanism. Unlike in tissue, the high resistance of the steel plate caused the lead core in the rear of the bullet to move forward perforating the central copper barrier behind the hollow point cavity. Thus, the peculiar fragment was created. The subsequent backwards acceleration of the ring-like fragment was probably caused by the partly elastic impact of the copper-alloy portion against the hard steel plate. Due to the perpendicular impact resulting in a centric and symmetrical deformation, the fragment moved in a direction exactly backwards along the original line of fire.

Mitochondrial HVS-I sequences from 10,365 subjects belonging to 56 populations/geographical regions of western Eurasia and northern Africa were first surveyed for the presence of the T-->C transition at nucleotide position 16298, a mutation which has previously been shown to characterize haplogroup V mtDNAs. All mtDNAs with this mutation were then screened for a number of diagnostic RFLP sites, revealing two major subsets of mtDNAs. One is haplogroup V proper, and the other has been termed "pre*V," since it predates V phylogenetically. The rather uncommon pre*V tends to be scattered throughout Europe (and northwestern Africa), whereas V attains two peaks of frequency: one situated in southwestern Europe and one in the Saami of northern Scandinavia. Geographical distributions and ages support the scenario that pre*V originated in Europe before the Last Glacial Maximum (LGM), whereas the more recently derived haplogroup V arose in a southwestern European refugium soon after the LGM. The arrival of V in eastern/central Europe, however, occurred much later, possibly with (post-)Neolithic contacts. The distribution of haplogroup V mtDNAs in modern European populations would thus, at least in part, reflect the pattern of postglacial human recolonization from that refugium, affecting even the Saami. Overall, the present study shows that the dissection of mtDNA variation into small and well-defined evolutionary units is an essential step in the identification of spatial frequency patterns. Mass screening of a few markers identified using complete mtDNA sequences promises to be an efficient strategy for inferring features of human prehistory.

Contact of a fired bullet with an intermediate target of sufficient resistance commonly causes the bullet to ricochet, fragment or perforate together with a deviation in trajectory. The transfer of intermediate target material to bullets and subsequent detection on the bullet surface after recovery from a bullet collector, were examined using a scanning electron microscope and an energy-dispersive X-ray spectrometer (SEM/EDS). A total of 76 gunshots (9 mm Luger FMJ RN bullets) were fired at various intermediate targets and at combinations of intermediate targets and tissue located in line. Elements already present on unfired bullets and elements from the bullet collector, the jacket, the charge and primer could be consistently detected as a "background". Abundant deposits of "fragile" (brittle) materials such as concrete, flat glass, asphalt and gypsum board could be visualised on every bullet by SEM. The transfer dynamics involved a direct imprint of target material on the bullet surface and thus preferential locations at the tip but also indirect deposition over the entire surface ("powder effect"). X-ray microanalysis demonstrated matching spectra of the elemental composition of these deposits and of the targets contacted. After perforation of "ductile" (flexible) materials such as wood and car body parts, the deposits on the bullets did not show characteristic spectra. If multi-layered car metal targets were hit, few and uncharacteristic fragments were scattered over the bullet surface and titanium indicative of paint-work could be determined on only a minority of bullets. The elemental composition of wood itself was heterogeneous but the fibrous morphology of the deposits was typical. The SEM/EDS findings in gunshots including subsequent perforation of tissue were similar. In particular, the trace evidence primarily transferred to the bullets was not eliminated by secondary contact and the determination of the fragile target materials was not affected. So when a person is killed or injured by a gunshot, the presence of a ricochet and the target material can be determined. This possibility needs to be considered before an evidential bullet is cleaned or contaminated.

If a pedestrian was run over by a car, the question can arise whether there was a preceding collision while the pedestrian was in an erect position. From a total of 53 selected autopsy reports, the findings associated with accidents known to involve running over in isolation (n=32) were compared to findings associated with a combined mechanism of a primary impact in an erect position and subsequent running over (n=21). Findings exclusively present in the combined group were wedge-shaped bone fractures ("Messerer"-wedges, 38%), glass fragment injuries (24%), traumatic amputations (10%), traces of car paint on the lower extremities (50%) and abrasions of the shoe soles (17%). These findings can be considered specific for a primary impact in an erect position. Fractures of the cervical and lumbar spine were present in the combined group in 33 and 17%, respectively. In contrast, in the run over group, there was only one case of fracture of the cervical and one of the lumbar spine and both cases involved direct contact with a car wheel. Fractures of the cervical and lumbar spine are, therefore, very indicative for a primary impact. "Bumper injuries", sacroiliac dislocations and fractures of the thoracic spine were approximately 2.5 times more common in the combined group than in the run over group. In the vast majority of cases, a clear differentiation between the two groups is, therefore, possible on the basis of the autopsy findings. This is especially relevant if an inspection of the car cannot be performed after a hit-and-run accident, which occurred in 26% of the cases in this study. In addition, the blood alcohol levels were higher in the run over group (mean=2.14g/l) as compared to the combined group (mean=1.53g/l).

A new design for high-throughput microfabricated capillary electrophoresis/electrospray mass spectrometry (CE/ ESI-MS) with automated sampling from a microwell plate is presented. The approach combines a sample-loading port, a separation channel, and a liquid junction, the latter for coupling the device to the MS with a miniaturized subatmospheric electrospray interface. The microdevice was attached to a polycarbonate manifold with external electrode reservoirs equipped for electrokinetic and pressure-fluid control. A computer-activated electropneumatic distributor was used for both sample loading from the microwell plate and washing of channels after each run. Removal of the electrodes and sample reservoirs from the microdevice structure significantly simplified the chip design and eliminated the need both for drilling access holes and for sample/buffer reservoirs. The external manifold also allowed the use of relatively large reservoirs that are necessary for extended time operation of the system. Initial results using this microfabricated system for the automated CE/ESI-MS analysis of peptides and protein digests are presented.

This paper describes the design and application of instrumentation for automated high-throughput infusion ESI-mass spectrometry. The approach, based on a subatmospheric ESI interface, allows sample introduction from a commercially available microtiter plate without the need for a separate fluid delivery system. The microtiter plate was placed vertically on a three-dimensional translation stage in front of the sampling ESI interface. A single, 7-cm, 20-microm-i.d. fused-silica capillary (total volume, 70 nL), with a tapered tip, served as a combination of sample delivery and spraying capillary. The tapered tip of the capillary was enclosed in a subatmospheric chamber attached in front of the orifice of the mass spectrometer. The sample aspiration rate (flow rate) was regulated by computer-controlled pneumatic valves, which allowed fast switching of the pressure in the subatmospheric ESI chamber. A flow-through wash device was positioned between the microtiter plate and the ESI interface. This design allowed alternate filling of the capillary with (a) sample from the wells and (b) wash solution from the wash device. Sample turnaround times of 10 s/sample, with a 120-nL sample consumption/analysis, and a duty cycle (percentage of total analysis time spent acquiring data) of 40% were achieved. The infusion system was demonstrated in the analysis of preparative HPLC fractions from a small molecule combinatorial library.

STR-based individualisation of biological deposits on bullets after perforation of tissue, can identify the person injured or killed by a particular bullet and comparison with the firearms used can identify the weapon and thus possibly the person who did the shooting. In this study, the effect of subsequent impacts on intermediate targets such as loss of cells was investigated by amplification of mitochondrial (mt) DNA. Bovine tissue was perforated and the 9 mm Luger FMJ bullets were recovered from the bullet collector. The mt cytochrome-b (cyt-b) gene could be amplified by the polymerase chain reaction (PCR) from 14 out of 15 bullets. Examination with a scanning electron microscope (SEM) and an energy-dispersive X-ray spectrometer (EDS) demonstrated the presence of minute dried tissue deposits on all bullets (n = 10) but was not able to establish preferential locations. In a series of 25 gunshots, various intermediate targets (glass, wood, car metal, gypsum board, asphalt) were perforated/impacted following perforation of tissue and the cyt-b gene could be typed from all bullets. It is concluded that subsequent impacts on intermediate targets do not eliminate enough biological deposits to render DNA analysis impossible and that the amplification of mtDNA is a useful additional method.

The diagnosis of a gunshot wound can be difficult especially if the morphology is not typical. In the case presented a neck injury was not recognised as a gunshot wound by several clinicians and radiologists although the bullet could be seen at the base of the patient's tongue and on all X-rays taken. This misinterpretation may have been caused by a "professional blinkers phenomenon".

A general method for noncompetitive immunoassay of small analytes using affinity probe capillary electrophoresis (APCE) is demonstrated using digoxin as a model analyte. A uniform immunoreagent was prepared from a single-chain antibody (scFv) gene specific for digoxin. Site-directed mutagenesis introduced a unique cysteine residue for uniform labeling with a thiol-reactive fluorochrome. After expression in E. coli, the scFv was purified by immobilized metal affinity chromatography (IMAC) using an added C-terminal 6-histidine sequence. The protein was renatured and labeled while immobilized on the IMAC resin. After 0.02-microm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 degrees C for at least 1 year. Three formats of APCE using the scFv reagent were explored. A "mix-and-inject" assay optimized for low detection limits demonstrated analysis of 10 pM digoxin in aqueous standard solutions in 10 min. A rapid mix-and-inject format in a short capillary allowed detection of 1 nM digoxin in 1 min. Digoxin samples in serum and urine were injected directly after 10-fold dilution. In combination with solid-phase extraction, 400 fM digoxin was detected in 1 mL of serum. Including solid-phase extraction, reproducibility was within 2.5%, and the linear range was 3 orders of magnitude. The strategy adopted in this paper should be of general use in the low-level analysis of small analytes.

An improved vacuum deposition interface for coupling capillary electrophoresis with MALDI-TOF MS has been developed. Liquid samples consisting of analyte and matrix were deposited on a moving tape in the evacuated source chamber of a TOF mass spectrometer, enabling 24 h of uninterrupted analysis. The vacuum deposition procedure was compared with the dried-droplet method, and it was found that vacuum deposition generated significantly more reproducible signal intensity, eliminating the need for "sweet spot" searching. A concentration detection limit in the low-nanomolar range has been achieved with a low-attomole amount of sample consumed per spectrum. In addition, ion suppression caused by hydrophobicity differences in the analytes was reduced. To minimize ion suppression further, separation prior to MALDI MS analysis was employed. The performance of capillary electrophoresis (CE)-MALDI-TOF MS using the vacuum deposition interface was evaluated with a peptide mixture injected at low-femtomole levels. All peptides were baseline resolved with separation efficiencies in the range of 250000-400000 plates/m (2-3-s band half-width), demonstrating the high separation efficiency of the CE-MALDI MS coupling. A fast (approximately 40 s) CE separation of a mixture of angiotensins was found to reduce significantly ion suppression and enable trace level detection. It was also shown, for the analysis of an enolase digest, that sequence coverage of 65% was obtained using CE separation compared to 52% using step-elution solid-phase extraction and 44% in the control experiment using an unseparated mixture.

The procedures used to sequence the human genome involve the electrophoretic separation of mixtures of dioxyribonucleic acid (DNA) fragments tagged with reporting groups, usually fluorescent dyes. Each fluorescent pulse which arrives from an optical detector corresponds to a nucleotide (base) in the DNA sequence, and the subsequent process of base detection is known as base calling. Generating longer and more accurate sequences in the base-calling process will reduce the high cost of DNA sequencing. This paper presents an automated base-calling algorithm, referred to as maximum-likelihood base caller (MLB), which is based on maximum likelihood equalization for digital communication channels. Based on 125 experimental datasets, MLB averaged up to 40% fewer errors than the widely used ABI base caller from the Applied Biosystems Division of PE Corporation. MLB's accuracy rivaled that of another well-known base caller, Phred, surpassing it on datasets with high background noise.

The basic principles of multichannel devices with an array of electrospray tips for high-throughput infusion electrospray ionization mass spectrometry (ESI-MS) have been developed. The prototype plastic devices were fabricated by casting from a solvent-resistant resin. The sample wells on the device were arranged in the format of the standard 96-microtiter well plate, with each sample well connected to an independent electrospray exit port via a microchannel with imbedded electrode. A second plastic plate with distribution microchannels was employed as a cover plate and pressure distributor. Nitrogen gas was used to pressurize individual wells for transport of sample into the electrospray exit port. The design of independent microchannels and electrospray exit ports allowed very high throughput and duty cycle, as well as elimination of any potential sample carryover. The device was placed on a computer-controlled translation stage for precise positioning of the electrospray exit ports in front of the mass spectrometer sampling orifice. High-throughput ESI-MS was demonstrated by analyzing 96 peptide samples in 480 s, corresponding to a potential throughput of 720 samples/h. As a model application, the device was used for the MS determination of inhibition constants of several inhibitors of HIV-1 protease.

A modular subatmospheric electrospray interface with fiber optic UV detection close to the electrospray tip was developed for coupling of microcolumn separation techniques with mass spectrometry. The interface was based on a liquid junction with a removable microelectrospray tip. The electrospray tip was enclosed in a subatmospheric chamber attached in front of the sampling orifice of the mass spectrometer. The inlet of the liquid junction was maintained at atmospheric pressure, and thus no pressure drop developed across the separation column. The flow rate of the electrosprayed liquid from the liquid junction reservoir was adjusted by the pressure in the electrospray chamber. In this approach, a continuous and stable electrospray could be achieved without the use of an external pump. Since the electrospray did not depend on fluid delivery from the separation column, coated capillaries without electroosmotic flow as well as capillaries with electroosmotic flow could be used for capillary electrophoresis. In addition, the interface was found to be effective with capillary liquid chromatography. The use of a fiber optic UV detector placed close to the exit of the separation column provided additional detection information and a simple means of troubleshooting. The interface did not significantly influence the quality of the separation, even with columns generating several hundred thousand theoretical plates. Peptide samples in the submicromolar concentration range were detected, corresponding to a limit of detection in the attomole range.

This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single-stranded M13 template.

A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High-efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis.

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 microm inner diameter (ID) were employed in CZE and 100 microm ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

The most common type of fatal pedestrian-car collision, i.e. a passenger car with a wedge or pontoon shaped front striking an erect adult with this front, was investigated. A reliable calculation of the impact velocity range by a technical expert and a comprehensive autopsy suitable for traffic accidents were performed in every case. A total of 47 fatalities form the material of this study and the impact velocities varied between 18 and 142 km/h. Primary and secondary injuries did not show a relationship to impact velocity. The occurrence of four types of indirect injuries revealed a clear relationship to impact velocity, i.e. spinal fractures, ruptures of the thoracic aorta, inguinal skin ruptures and dismemberment of the body. Important parameters such as the type of car, impact velocity range and indirect injuries are listed for each individual case. Because of the limited number of cases, the impact velocity ranges (3-30 km/h) instead of mean values were considered. A cautious interpretation of the data can be summarised in the following conclusions: If there is no spinal fracture, the velocity was below 70 km/h and probably below 50 km/h. Aortic and inguinal skin ruptures are always present if the velocity was above 100 km/h but never occurred below 50-60 km/h. If dismemberment occurs, the velocity was above 90 km/h. Consequently, an estimation of the impact velocity from the presence or absence of indirect injuries is possible in pedestrian-car collisions of the type examined. However, the selection criteria applied in this study and additional parameters influencing the collision dynamics have to be considered carefully.

This work demonstrates the use of affinity probe capillary electrophoresis (APCE) in the quantitative analysis of drugs in biological fluids at the low pM level. The interaction of human carbonic anhydrase II (HCAII) with the glaucoma drug dorzolamide (Dz) was chosen as a model system. HCAII was labeled at its single cysteine residue using a thiol-specific fluorescein reagent. The peak area of HCAII complexed with the tight-binding drug Dz provided a direct assay of the drug concentration in solution. A charged competitive ligand added to the running buffer was employed in APCE to distinguish Dz-bound from free forms of the HCAII. Using laser-induced fluorescence (LIF), the Dz detection limit was 16.5 pM in aqueous solution and 62.5 pM in both urine and plasma. Normalized peak area reproducibility of the drug was within 3.4% RSD. Each analysis was completed within 10 min, including incubation, and consumed only 0.3 pmol of labeled protein. The APCE approach provides an effective method for trace level detection of drugs in biological matrices.

A total of 65 consecutive cases of suicide by sharp force were investigated by evaluating the autopsy and prosecution department records. Suicides constituted 17% of all fatalities from sharp force autopsied between 1967 and 1996. Young males and persons with a psychiatric history predominated among the persons who chose this "hard" method of suicide. The most common implements used were knives (62%) and razor blades (15%). Cutting injuries in isolation were present in 26, stab injuries in isolation in 24 and a combination of both in 15 fatalities. The number of injuries per case varied from 1 to 37 but 1/3 showed one injury. More than 85% of the cutting injuries were located at the wrist, elbow crease or neck whereas 79% of the stab injuries involved the ventral aspect of the trunk. Perforation of clothing was present in 16 (52%) out of 31 stab injuries to the trunk. Injuries to more than one body region were observed in 34 (52%) cases. Tentative marks were present in 50 (77%) fatalities and the number varied from 1 to 60 per case. Superficial incisions of the fingers were found in 15% with razor blades constituting the weapon in half of these cases. Deviations from these typical patterns occurred not infrequently. The utter determination of the victim to carry it through or the use of unusual weapons resulted in a few bizarre cases which are outlined briefly.

A corpse of a female was found in her apartment in a state of advanced putrefaction. Both hands were amputated, the external genitals were excised and the body parts had been removed from the scene. The subsequent investigations proved that the body parts had been severed post mortem. The cause of death was determined to be manual strangulation. A 33-year-old man later confessed that he had strangled the victim 9 days prior to discovery of the body and that he had had sexual intercourse post mortem. According to the confession, the rational motive for the subsequent mutilation was to eliminate biological stains (e.g. semen inside the vagina, epithelial cells below the fingernails from scratching) suitable for forensic DNA analysis. This constitutes a new type of defensive mutilation intended to prevent the identification of the perpetrator. An increase in the occurrence would be detrimental to the elucidation rate of homicides: in a total of 171 homicides investigated at this institute, DNA analysis of biological stains gave reliable evidence in 45 cases.

This paper describes the analysis of recombinant human deoxyribonuclease (rhDNAse), an acidic and complex phosphoglycoprotein, by capillary zone electrophoresis (CZE). Separation performance was found to be dramatically improved by the addition of calcium ions to the CZE running buffer, due to the influence of calcium binding on the charge and the electrophoretic behavior of rhDNAse. The pH dependent calcium binding effects on the electrophoretic separation were demonstrated at both acidic and basic pH, resulting in a two-dimensional (pH 4.8 and 8.0) calcium aided analysis that achieved multipeak resolution of the complex, glycosylation based, charge microheterogeneity of rhDNAse. Two-dimensional investigation of neuraminidase- and alkaline phosphatase-digested protein further demonstrated that the acidic pH resolved acidic charge heterogeneity and that the basic pH discriminated neutral heterogeneity. This work demonstrates the resolving power of CZE for the analysis of a complex microheterogeneous glycoprotein, and emphasizes the importance of employing multiple separation conditions in accordance with known structural characteristics of the protein.

Two fundamental approaches for the coupling of microfabricated devices to electrospray mass spectrometry (ESI-MS) have been developed and evaluated. The microdevices, designed for electrophoretic separation, were constructed from glass by standard photolithographic/wet chemical etching techniques. Both approaches integrated sample inlet ports, preconcentration sample loops, the separation channel, and a port for ESI coupling. In one design, a modular, reusable microdevice was coupled to an external subatmospheric electrospray interface using a liquid junction and a fused silica transfer capillary. The transfer capillary allowed the use of an independent electrospray interface as well as fiber optic UV detection. In the second design, a miniaturized pneumatic nebulizer was fabricated as an integral part of the chip, resulting in a very simple device. The on-chip pneumatic nebulizer provided control of the flow of the electrosprayed liquid and minimized the dead volume associated with droplet formation at the electrospray exit port. Thus, the microdevice substituted for a capillary electrophoresis instrument and an electrospray interface--traditionally two independent components. This type of microdevice is simple to fabricate and may thus be developed either as a part of a reusable system or as a disposable cartridge. Both devices were tested on CE separations of angiotensin peptides and a cytochrome c tryptic digest. Several electrolyte systems including a transient isotachophoretic preconcentration step were tested for separation and analysis by an ion trap mass spectrometer.

Constant denaturant capillary electrophoresis (CDCE) permits high-resolution separation of single-base variations occurring in an approximately 100 bp isomelting DNA sequence based on their differential melting temperatures. By coupling CDCE for highly efficient enrichment of mutants with high-fidelity polymerase chain reaction (hifi PCR), we have developed an analytical approach to detecting point mutations at frequencies equal to or greater than 10(-6) in human genomic DNA. In this article, we present several applications of this approach in human genetic studies. We have measured the point mutational spectra of a 100 bp mitochondrial DNA sequence in human tissues and cultured cells. The observations have led to the conclusion that the primary causes of mutation in human mitochondrial DNA are spontaneous in origin. In the course of studying the mitochondrial somatic mutations, we have also identified several nuclear pseudogenes homologous to the analyzed mitochondrial DNA fragment. Recently, through developments of the means to isolate the desired target sequences from bulk genomic DNA and to increase the loading capacity of CDCE, we have extended the CDCE/hifi PCR approach to study a chemically induced mutational spectrum in a single-copy nuclear sequence. Future applications of the CDCE/hifi PCR approach to human genetic analysis include studies of somatic mitochondrial mutations with respect to aging, measurement of mutational spectra of nuclear genes in healthy human tissues and population screening for disease-associated single nucleotide polymorphisms (SNPs) in large pooled samples.

Cases of suicide from sharp pointed weapons (n = 12) witnessed by one or more persons are reported with regard to the potential for physical activity. One case each involved the ulnar artery, the great saphenous vein and the periphery of the lung and liver and the physical activity following these injuries lasted for several hours. In one case, the left carotid and vertebral arteries were transected and the physical activity lasted for approximately 10 s. An extraordinary case involved a protracted incapacitation due to heart tamponade from a small myocardial injury caused by a cannula. In the remaining seven cases, a stab wound to the heart was present. With regard to the physical activity, a long-term group (2-10 min, n = 4) can be differentiated from a short-term group (approximately 10 s, n = 2) and one case of immediate incapacitation. The size of the myocardial perforation was 7-10 mm in length in the long-term group compared to 1.4-2 cm in the short-term group. So small perforations of the heart or incisions of the carotid artery offer a potential for considerable physical activity. Large perforations of the heart or a transection of the carotid and vertebral arteries can result in short-term activity.

Four cases of hand grenade detonations in civilian life with homicidal intent are reported. The detonations always occurred in close proximity to the victims but there were only two fatalities out of seven victims directly attributable to the hand grenades. The relatively low mortality rate results from the ballistic characteristics of hand grenade missiles such as low mass and sectional density. This leads to rapid deceleration and thus to a poor penetration capacity of intermediate targets and tissue. Window glass at a distance of 2 m was not perforated in one case and the vast majority of fragment wounds showed a short wound tract of small diameter. The potential for physical activity can be unaffected even after a detonation inside a car. However, body parts almost in contact with the hand grenade, such as in a struggle, suffer large lacerations and comminuted fractures. At a distance, the chance of striking a vulnerable body region is increased by the multiple missiles but the wounding potential clearly decreases with increasing distance due to rapid deceleration and decreasing fragment cloud density. The effective range of detonation shock waves from hand grenades is very short and can only contribute to wounding in close-to-contact detonations. The forensic reconstruction is based on the directed radial character of the forces generated and on the sharply decreasing intensity of these forces with increasing distance from the detonation.

A man suffered a fatal injury from a self-inflicted accident while handling a special type of knife. A spring in the shaft of the knife accelerated the blade, which perforated the orbital cavity and the frontal lobe at the right side. Death was due to central disregulation. The initial velocity of the blade was measured to be 15 m/s. In a total of 20 experimental shots to a fresh pig cadaver, the blade always penetrated the skin and 5-10 cm of soft tissue as long as the distance did not exceed 1 m. Thin layers of bone were also perforated. The free flight of the blade did not remain stable if the distance was more than 1 m, which resulted in superficial wounds only. So this unusual construction resembling a knife can be considered an effective combat weapon for close range fighting instead of a tool.

Three suicides and one homicide from black powder muzzle loading handguns are reported and the muzzle velocities of two weapons are recorded. The fatal head shots caused wide wound tracts traversing the brains and intracerebral haemorrhages remote from the tract were present in every case. The skulls showed considerable fractures which were pronounced in the cases of contact shots but were also present when the range of fire was 5 m. These extensive injuries from soft lead spheres with muzzle velocities of only approximately 200 m/s are attributed to the expansion of the spheres. The regularly occurring deformation resulted in 13-16 mm calibre missiles in the cases of .44 spheres. The special features of black powder such as incomplete and slow combustion resulted in intense soot deposits in the vicinity of the entrance wound and in long ranges the gunshot residues travelled. In contact shots, large pocket-like underminings even in deeper tissue layers, abundant soot along the trajectory and skin burns were observed.

In this work, a new interface for continuous on-line MALDI-TOF MS is presented. The sample, mixed with a suitable matrix, was transported into the evacuated source chamber of the mass spectrometer at liquid flow rates of 100-400 nL/min. The liquid sample matrix was deposited on a rotating quartz wheel and transported to the repeller, where laser desorption took place. Rapid evaporation of the solvent (water or methanol) on the surface of the wheel resulted in formation of a thin, approximately 50-micron-wide, sample trace. Scanning electron microscopic photographs of the vacuum-dried trace revealed the deposited material to consist of an amorphous film. Furthermore, sample uniformity along the trace, in conjunction with its narrow width, resulted in excellent signal reproducibility, with detection limits in the attomole range. The interface permitted the on-line coupling of microcolumn separation techniques with MALDI MS, as demonstrated in the capillary electrophoresis MALDI-TOF MS analysis of a 12-peptide mixture. The approach offers the potential for rapid separation and trace analysis of complex mixtures.

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.

Long, accurate reads are an important factor for high-throughput de novo DNA sequencing. In previous work from this laboratory, a separation matrix of high-weight-average molecular mass (HMM) linear polyacrylamide (LPA) at a concentration of 2% (w/w) was used to separate 1000 bases of DNA sequence in 80 min with an accuracy close to 97% (Carrilho, E.; et al. Anal. Chem. 1996, 68, 3305-3313). In the present work, significantly improved speed and sequencing accuracy have been achieved by further optimization of factors affecting electrophoretic separation and data processing. A replaceable matrix containing a mixture of 2.0% (w/w) HMM (9 MDa) and 0.5% (w/w) low-weight-average molecular mass (50 kDa) LPA was employed to enhance the separation of DNA sequencing fragments in CE. Experimental conditions, such as electric field strength and column temperature, as well as internal diameter of the capillary column, have been optimized for this mixed separation matrix. Under these conditions, in combination with energy-transfer (BigDye) dye-labeled primers for high signal-to-noise ratio and a newly developed expert system for base calling, the electrophoretic separation of 1000 DNA sequencing fragments of both standard (M13mp18) and cloned single-stranded templates from human chromosome 17 could be routinely achieved in less than 55 min, with a base-calling accuracy between 98 and 99%. Identical read length, accuracy, and migration time were achieved in more than 300 consecutive runs in a single column.

A multicentre study involving 36 out of 38 German medico-legal Institutes discovered 784 non-natural fatalities among 78,000 external examinations before cremation (1%) in 1995. These included 169 accidents, 16 suicides, 3 homicides and 589 deaths in connection with medical treatment. In the remaining 7 cases, a belated differentiation between homicide and accident was not successful. A linear projection results in 1980 non-natural fatalities among all 197,000 external examinations before cremation. This number includes 8 homicides but due to considerable inhomogeneities, a number of 40 homicides appears to be realistic. Consequently, abolishing the mandatory external examination before cremation would be equivalent to an official renouncement of solving relevant non-natural deaths. In case of a thorough external examination, a total of 18,000 non-natural fatalities including approximately 180 homicides could be detected per year during the first external examinations (n = 885,000).

A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.

In the previous paper, a sample cleanup procedure for DNA sequencing reaction products was developed, in which template DNA was removed by ultrafiltration and the total concentration of salts (chloride and di- and deoxynucleotides) was decreased below 10 microM using gel filtration. In this paper, a quantitative study of the effects of these sample solution components on the injected amount and separation efficiency of the sequencing fragments in capillary electrophoresis is presented. The presence of chloride and deoxynucleotides in a total concentration above 10 microM in the sample solution significantly decreased the amount of DNA sequencing fragments injected into the capillary column. However, the separation efficiency was not affected upon increasing the amount of salt. On the other hand, in the presence of only 0.1 microgram of template in the sample (one-third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentration (approximately 5 microM), the separation efficiency decreased by 70%, and the injected amount of DNA sequencing fragments was 40% lower compared to the sample cleaned by the new purification method. The deleterious effect of template DNA on the separation of sequencing fragments was suppressed in the presence of salt in a concentration above 100 microM in the sample solution. Separately, it was found that both the electric field strength and duration of injection affected the resolution of DNA sequencing fragments when the cleaned up sample solution was used. Separation efficiencies of 15 x 10(6) theoretical plates/m were achieved when the sample was loaded at low electric field, e.g., 25 V/cm for 80 s or less. The results demonstrate that the sample solution components (chloride, deoxynucleotides, template DNA) and injection conditions must be controlled to achieve high performance and rugged DNA sequencing analysis.

On-line affinity capillary electrophoresis-electrospray ionization-mass spectrometry (ACE-MS) was used for the simultaneous measurement of multiple binding constants of an all-D-tetrapeptide library to the model receptor, vancomycin. Determination of Kd values for the 19 peptides of the form Fmoc-DXYA is demonstrated. The data are compared with the results obtained for individual compounds using ACE-UV, and good correlation between the two detection methods is shown. Simultaneous determination of multiple Kd values by ACE-MS is achieved in one set of experiments, whereas only one Kd value can be obtained by ACE-UV during the same time. ACE-MS measures multiple binding constants in solution in a fast and reliable manner using femtomole amounts of samples.