On-line High-performance Liquid Chromatography Research Articles

Case study and application of process analytical technology (PAT) towards bioprocessing: II. Use of ultra‐performance liquid chromatography (UPLC) for making real‐time pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. Two of the key goals that have been outlined for PAT are "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". Recently, we have been examining the feasibility of applying different analytical tools for designing PAT applications for bioprocessing. We have previously shown that a commercially available online high performance liquid chromatography (HPLC) system can be used for analysis that can facilitate real-time decisions for column pooling based on product quality attributes (Rathore et al., 2008). In this article we test the feasibility of using a commercially available ultra- performance liquid chromatography (UPLC) system for real-time pooling of process chromatography columns. It is demonstrated that the UPLC system offers a feasible approach and meets the requirements of a PAT application. While the application presented here is of a reversed phase assay, the approach and the hardware can be easily applied to other modes of liquid chromatography.

Characterization of polyprenylated xanthones in Garcinia xipshuanbannaensis using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

A reliable and sensitive on-line high-performance liquid chromatography (HPLC) coupled with electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS/MS) method has been optimized and established for the analysis of polyprenylated xanthones in the plant Garcinia xipshuanbannaensis. Collision induced MS/MS techniques were used to fragment the precursor molecular ions and MS/MS/MS techniques based on cone voltage fragmentation were used to further break down the resulting product ions sequentially. It was found that Retro–Diels–Alder rearrangement occurred from the xanthone skeleton in the MS/MS/MS process and produced characteristic fragment ions, which are useful for differentiating some positional isomers containing the prenyl unit on the A ring or B ring. Complementary fragmentation information, for instance the successive loss of prenyl residues, is also valuable for the identification of this class of xanthones. Under optimized HPLC–MS/MS/MS method, a total of 15 prenylated xanthones could be separated within 10 min. This method also provided information about the molecular formula of a precursor molecule and its fragments, which could be used for dereplication of known or likely new prenylated xanthones in Garcinia plants before the purification and structural elucidation process.

Analysis of flavonol glycoside isomers from leaves of Maytenus ilicifolia by offline and online high performance liquid chromatography–electrospray mass spectrometry

Flavonol glycosides present in leaves of Maytenus ilicifolia, were examined after fractionation on silica-gel column. Flavonol mono-, di-, tri-, and tetraglycosides, containing kaempferol, quercetin or myricetin were identified by offline electrospray mass spectrometry. Increasing the cone energy induced to adducts variation, from H + to Na +. Protonated ions were characteristically fragmented by sequentially removing the monosaccharide residues, whereas in the sodiated ions, the aglycone was firstly removed. Online high performance liquid chromatography–mass spectrometry, with simple gradients of water, acetonitrile and acetic acid indicated the presence of several isomers, which were further identified by gas chromatography–mass spectrometry as containing galactose or glucose.

On-line liquid chromatography–mass spectrometry with dilution line to achieve large volume urine injection for the improvement of sensitivity

A unique automated on-line high-performance liquid chromatography–mass spectrometry (LC–MS) method was developed allowing injection of a large volume of urine for the improvement of sensitivity using estrogens as analytes. The urine sample was precipitated by acetonitrile (sample:acetonitrile, 1:2, v/v) to remove most proteins. Then the supernatant was directly delivered at 0.3 mL/min by a pump, which broke through the limit of autosampler with fixed loop. Then the sample was loaded onto a restricted access media (RAM) pre-column after on-line dilution with NH 4Ac solution which was delivered by another pump under high flow-rate. After the addition of on-line dilution line, the content of organic solvent in the sample aliquot was sharply decreased so that the analytes were effectively trapped by pre-column. In addition, another 6-port valve was added to selectively deliver the effluent containing analytes into MS, which minimized the pollution of the MS and matrix effect. After optimization of the condition of extraction method, 3 mL sample (containing 1 mL urine) could be successfully enriched. The injection volume increased 10 times compared with conventional on-line LC–MS methods for biofluid analysis, significantly improving sensitivity. Moreover, no obvious increase of column pressure was observed after 300 injections. The method was validated by spiking urine. Linearity was determined by sample which was in the range of 1–500 ng/mL. The process efficiency ranged from 70.2 to 106% with RSDs less than 15% with the exception of a few analytes. The limit of detection (LOD) and quantification (LOQ) ranged from 0.3 to 2 ng/mL and 1 to 8 ng/mL, respectively, corresponding a decrease of 1–2 orders of magnitude compared with those of off-line and on-line methods.

Assessment of total arsenic and arsenic species stability in alga samples and their aqueous extracts

In order to achieve reliable information on speciation analysis, it is necessary to assess previously the species stability in the sample to analyse. Furthermore, in those cases where the sample treatment for species extraction is time-consuming, an assessment of the species integrity in the extracts is of paramount importance. Thus, the present paper reports total arsenic and arsenic species stability in alga samples ( Sargassum fulvellum and Hizikia fusiformis), as well as in their aqueous extracts, which were stored in amber glass and polystyrene containers at different temperatures. Total arsenic determination was carried out by inductively coupled plasma atomic emission spectroscopy (ICP-AES), after sample acid digestion in a microwave oven, while arsenic speciation was conducted by anion exchange high performance liquid chromatography on-line coupled to ICP-AES, with and without sample introduction by hydride generation (HPLC-ICP-AES and HPLC-HG-ICP-AES), after aqueous microwave-assisted extraction. The results obtained for solid alga samples showed that total arsenic (for Hijiki alga) and arsenic species present (As(V) for Hijiki and NIES No. 9 Sargasso) are stable for at least 12 months when samples are stored in polystyrene containers at +20 °C. On the other hand, a different behaviour was observed in the stability of total arsenic and As(V) species in aqueous extracts for both samples, being the best storage conditions for Sargasso extracts a temperature of −18 °C and polystyrene containers, under which they are stable for at least 15 days, while Hijiki extracts must be stored in polystyrene containers at +4 °C in order to ensure the stability for 10 days.

On‐line coupling of an ultraviolet titanium dioxide film reactor with a liquid chromatography/hydride generation/inductively coupled plasma mass spectrometry system for continuous determination of dynamic variation of hydride‐ and nonhydride‐forming arsenic species in very small microdialysate samples

We describe a method for continuously monitoring both hydride- and nonhydride-forming arsenic species in 10-microL microdialysate samples by coupling together on-line high-performance liquid chromatography (HPLC), a post-column UV/TiO2 film reactor, and hydride generation (HG) inductively coupled plasma mass spectrometry (ICP-MS). To maximize the signal intensities of the desired arsenic species, we optimized the photocatalytic oxidation efficiency of the analyte species and used a rapid on-line pre-reduction process to convert the oxidized species into As(III) prior to HG-ICP-MS determination. The UV/nano-TiO2 film reactor was manufactured by coating nano-TiO2 onto the interior of a glass tube. Impregnation and sol-gel methods were employed to deposit the TiO2 films, and their effectiveness for the oxidation of organic arsenicals was compared. To enhance the decomposition efficiency of organic arsenicals, we investigated the effects of the acidity and the composition of the column effluent. Because of the improved HG efficiency toward the tested arsenicals and the adoption of a segmented flow technique to retain the peak resolution in our on-line LC-UV/nano-TiO2 film reactor-HG-ICP-MS instrument, the detection limits for arseneous acid [As(III)], monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenic acid [As(V)], and arsenobetaine (AsB) were all in the submicrogram-per-liter range (based on 3 sigma) for 10-microL injections. A series of validation experiments--analyses of certified reference urine and rabbit serum samples--indicated that these methods can be applied satisfactorily to the continuous determination of As(III), MMA, DMA, As(V), and AsB in blood and in the extracellular space of target organs.

Case study and application of process analytical technology (PAT) towards bioprocessing: Use of on‐line high‐performance liquid chromatography (HPLC) for making real‐time pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. This paper presents a PAT application for one of the most commonly used unit operation in bioprocessing, namely liquid chromatography. Feasibility of using a commercially available online-high performance liquid chromatography (HPLC) system for real-time pooling of process chromatography column is examined. Further, experimental data from the feasibility studies are modeled and predictions of the model are compared to actual experimental data. It is found that indeed for the application under consideration, the online-HPLC offers a feasible approach for analysis that can facilitate real-time decisions for column pooling based on product quality attributes. It is shown that implementing this analytical scheme allows us to meet two of the key goals that have been outlined for PAT, that is, "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." Finally, the implications of implementing such a PAT application in a manufacturing environment are discussed. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis.

Development and characterization of β-secretase monolithic micro-immobilized enzyme reactor for on-line high-performance liquid chromatography studies

β-Site APP cleavage enzyme 1 (BACE-1) is a transmembrane aspartyl protease that cleaves the amyloid-β precursor protein (APP), which is abundant in neurons. BACE-1 is required for the generation of amyloid-β (Abeta) peptides implicated in the pathogenesis of Alzheimer's disease (AD). It is widely believed that halting the production of Abeta peptide, by inhibition of BACE-1, is an attractive therapeutic modality for the treatment of Alzheimer's disease. BACE-1 has never been immobilized before. In the present study, for the first time, human recombinant β-secretase micro-immobilised enzyme reactor (hrBACE-1-micro-IMER) was prepared by using an in situ immobilisation procedure on an ethylendiamine monolithic convective interaction media (EDA-CIM) disk. The activity and kinetic parameters of the hrBACE-1-micro-IMER were investigated by insertion in a HPLC system with fluorescent and mass detection. The micro-IMER was characterized in terms of units of immobilised hrBACE-1 and best mobile phase conditions for activity, by using as substrate casein-FITC and JMV2236, a peptide mimicking the Swedish-mutated APP (amyloid precursor protein) sequence. The characterization of the hrBACE-1-micro-IMER in terms of number of enzymatic active units after covalent linking to the solid matrix was performed by using the JMV2236 peptide as substrate in a HPLC-MS system. JMV2236 was injected into the hrBACE-1-micro-IMER and enzymatically cleaved; the product of the enzymatic cleavage and the remaining non-cleaved substrate were collected on a C18 column trap and switched to the LC-electrospray ionization MS system for kinetic constants determination. Inhibition studies were carried out. The effect of donepezil and pepstatin A, as BACE-1 inhibitors, was evaluated by simultaneous injection of the compounds with the peptidic substrate. The relative IC 50 values were found in agreement with that derived by the conventional fluorescence method, confirming the applicability of this new IMER for on-line inhibition studies. The main advantages of the hrBACE-1-micro-IMER approach over the conventional methods were found to be the increased enzyme efficiency, stability and the decreased time of analysis.

Simultaneous analysis of multiple bioactive constituents in Rheum tanguticum Maxim. ex Balf. by high‐performance liquid chromatography coupled to tandem mass spectrometry

A simple, rapid method was developed using on-line high-performance liquid chromatography (HPLC)-diode array detection (DAD)/electrospray ionization mass spectrometry (ESI-MS) to simultaneously analyze multiple bioactive constituents in the extract of Rheum tanguticum Maxim. ex Balf. (Dahuang), a traditional Chinese medicine (TCM). Many bioactive constituents gave prominent [M-H]- ions in the negative ion ESI mass spectra. Among them, 41 different constituents including 16 anthraquinone derivatives, 7 phenylbutanone glucopyranosides, 4 stilbenes and 14 tannins were unambiguously identified or tentatively characterized based on their retention times, UV spectra and mass spectra in comparison with the data from standards or references. Meanwhile, some principles of fragmentation behavior for different types of constituents were proposed, which could contribute to the elucidation of these constituents in Rheum tanguticum.

Chromatographic fingerprint analysis of Cephalotaxus sinensis from various sources by high-performance liquid chromatography–diodearray detection–electrospray ionization-tandem mass spectrometry

Selective and efficient analytical methods are required not only for quality assurance but also for authentication of Chinese herbal medicine. A simple, rapid and valid fingerprint method has been first carried out for the quality control of Cephalotaxus sinensis by using high-performance liquid chromatography (HPLC) coupled with photodiode array detection (DAD) and electrospray ionisation-tandem mass spectrometry (ESI-MS). The characteristic analytical fingerprints of this plant extract showed 18 common peaks, and out of these, 10 compounds involving 2 new compounds were identified by comparing the retention time, UV and ESI-MS/MS spectrum of each standard with those of each peak separated by on-line HPLC–DAD–MS/MS. Moreover, the effects of collecting locations, harvesting time, storage time, drying methods, and medicinal portions on herbal chromatographic fingerprints were examined by similarity analysis and principal component analysis (PCA) along with markers. Using the reference fingerprint along with markers, the best harvesting time, cultivation location and medicinal part were determined. The results obtained suggest that the chromatographic fingerprint combining similarity evaluation and PCA along with markers or pharmacologically active constituents can efficiently identify raw herb of Cephalotaxus sinensis from different sources, which provide helpful clues to the study of plant's secondary metabolites and benefit quality control.

Evaluation of extraction methods for arsenic speciation in polluted soil and rotten ore by HPLC-HG-AFS analysis

Three extraction systems including shaking, ultrasonic and microwave-assisted extraction were evaluated. Water and phosphate buffer were tested for the extraction of arsenic compounds in polluted soil, describing the water-soluble or plant-available fraction. The stabilities and recoveries of various arsenic species indicated that no obvious changes of species occurred during the extraction process. The raw extracts were cleaned up by C18 cartridge before analysis. Having optimized the extraction conditions, the arsenic species in polluted soil and ore from the different pollution sources were extracted by microwave-assisted extraction with 0.5 M phosphate buffer as extractant. Arsenic species were quantitatively determined by high performance liquid chromatography on-line coupled with hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS). As(III) and As(V) were the major arsenic species in the polluted soil samples resulting from irrigation by waste water. AsV was the only form found in the rotten ore sampled in mining area. During the extraction process, the recoveries of spiked As(III), As(V), DMA(V) and MMA(V) were 85.4 ± 7.2%, 80.2 ± 6.7%, 101.6 ± 6.7% and 98.8 ± 9.1%, respectively, showing that most water-soluble arsenic could be measured.

Arsenic removal from rice by washing and cooking with water

Two Hungarian and one Chinese rice samples were selected in order to establish the extractable arsenic content by washing and cooking in water in a ratio of 6:1, water:rice (cm 3:g) by inductively coupled plasma sector field mass spectrometry (ICP-SF-MS). Total arsenic concentration of the Zhenshan 97, Risabell and Ko˝röstáj-333 samples were 171.3 ± 7.1 ng g −1, 116.0 ± 3.7 ng g −1 and 139.0 ± 6.1 ng g −1, respectively, which did not exceed the toxic limits established for As in Hungary (0.3 μg g −1) or in China (0.7 μg g −1). The predominant chemical form of As in the raw rice samples determined by on-line high performance liquid chromatography and ICP-MS was arsenite. Moreover, enzymatic hydrolysis with α-amylase + protease and microprobe focused sonication proved that arsenite could be removed in the highest extent by washing and cooking, meanwhile the main As form remaining in the cooked rice was As(V). Thus, it is recommended to prepare rice-containing dishes in abundant water, which should be discarded after washing and cooking. The results were validated with a NIST SRM 1568a.

Characterization of the components present in the active fractions of health gingers ( Curcuma xanthorrhiza and Zingiber zerumbet) by HPLC–DAD–ESIMS

Curcuma xanthorrhiza and Zingiber zerumbet are two of the most commonly used ingredients in Indo-Malaysian traditional medicines, health supplements and tonics. Recently, a number of products derived from the aqueous extracts of these species have appeared in the market in the form of spray-dried powder packed in sachet or bottle. On-line high performance liquid chromatography, coupled with diode array detection and electrospray ion trap tandem mass spectroscopy (HPLC–DAD–ESI–MS n), was used to analyze the components in the antioxidant-active fractions from the rhizomes of these species. Three components were identified from C. xanthorrhiza, including bisdemethoxycurcumin ( 1), demethoxycurcumin ( 2) and curcumin ( 3). The active fraction from Z. zerumbet consisted of five components, including kaempferol 3- O-rhamnoside ( 4), compound 5 [kaempferol 3- O-(2″- O-acetyl)rhamnoside ( 5a) or kaempferol 3- O-(3″- O-acetyl)rhamnoside ( 5b)], kaempferol 3- O-(4″- O-acetyl)rhamnoside ( 6), kaempferol 3- O-(3″,4″- O-diacetyl)rhamnoside ( 7) and kaempferol 3- O-(2″,4″- O-diacetyl)rhamnoside ( 8). To confirm their identities, the components from Z. zerumbet were isolated conventionally and were analyzed by spectroscopic techniques as well as by comparison with literature data.

Pharmacokinetics of Taurolidine following Repeated Intravenous Infusions Measured by HPLC-ESI-MS/MS of the Derivatives Taurultame and Taurinamide in Glioblastoma Patients

Taurolidine is known to have antimicrobial activity. Furthermore, at lower concentrations, it has been found to exert a selective antineoplastic effect in vitro and in vivo. The aim of this study was to investigate the pharmacokinetics of taurolidine in vivo following repeated intravenous infusion in a schedule used for the treatment of glioblastoma. As a prerequisite, the pharmacokinetics of taurolidine in human blood plasma and whole blood in vitro was investigated. The pharmacokinetics of taurolidine and its derivatives taurultame and taurinamide were investigated in human blood plasma and in whole blood in vitro using blood from a healthy male volunteer. During repeated intravenous infusion therapy with taurolidine, plasma samples were taken every hour for a period of 13 hours per day in seven patients (three male, four female; mean age 48.4 +/- 12.8 years, range 27-66 years) with a glioblastoma. Following dansyl derivatisation, the concentrations of taurultame and taurinamide were determined using a new method based on high-performance liquid chromatography (HPLC) online coupled to electrospray ionisation tandem mass spectrometry (ESI-MS/MS) in the multiple reaction monitoring mode. Under the experimental conditions used, taurolidine could not be determined directly and was back-calculated from the taurultame and taurinamide values. The new HPLC-ESI-MS/MS method demonstrated high accuracy and reproducibility. In vitro plasma concentrations of taurultame and taurinamide remained constant over the incubation period. In whole blood in vitro, a time-dependent formation of taurinamide was observed. At the start of the incubation, the taurultame-taurinamide ratio (TTR) was 0.95 at an initial taurolidine concentration of 50 microg/mL, and 1.69 at 100 microg/mL. The concentration of taurultame decreased at the same rate as the taurinamide concentration increased, showing logarithmic kinetics. The calculated taurolidine concentration remained largely constant over the 6-hour incubation period. During repeated infusions in patients, calculated plasma concentrations of taurolidine showed a strong increase after the start of each infusion and continued to increase until the end of infusion, followed by a rapid decline. The TTR was found to fluctuate between 0.1 and 0.3, depending on the relation to the previous or next infusion period. The volume of distribution was markedly higher for taurolidine, taurultame and taurinamide than the plasma volume. Taurolidine displayed a stable pattern of derivatives in plasma in vitro, whereas in whole blood, a time- and concentration-dependent conversion was apparent. In patients, the calculated average taurolidine plasma concentration, achieved with the repeated infusion regimen, was in the antineoplastic-effective concentration range. The tissue concentrations of taurolidine and taurultame are expected to be higher than the plasma concentrations, taking into account the calculated volumes of distribution. Repeated infusion of taurolidine is the therapeutically adequate mode of administration for the indication of glioblastoma.

Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products

An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin- O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent metabolite resorufin. The system was tested with three P450 1A2 inhibitors, for which minimum detectable amounts (MDA) ranging from 0.7 to 9.5 ng were obtained. Analysis of a kava kava and a basil extract showed that the on-line system is applicable to complex mixtures, since in both extracts, peaks with P450 1A2 inhibiting activity were observed.

On-line HPLC combined with multivariate statistical process control for the monitoring of reactions

On-line high performance liquid chromatography is used to monitor a steady state reaction over 35.2 h, with 197 chromatograms recorded as the reaction progresses. For each chromatogram, peaks are detected, baseline corrected, aligned and integrated to provide a peak table consisting of the intensities of 19 peaks, two corresponding to the reactants, one to the product and one to the solvent, the remaining being impurities, by-products or intermediates. D-charts and Q-charts from multivariate statistical process control are applied to the data to determine which samples are out of control and also provide diagnostic insight into why these samples are problematic. The D-chart is best at looking at overall performance issues such as problems with mixing or difficulties with instrument operation, whereas the Q-charts are best at detecting impurities during the reaction.

Identification of the novel thio‐arsenosugars DMThioAsSugarCarboxyl, DMThioAsSugarCarbamate and DMThioAsSugarAdenine in extracts of giant clam tissues by high‐performance liquid chromatography online with electrospray tandem mass spectrometry

Recently, the identification of four thio-arsenosugars in extracts of marine samples was reported. The biosynthesis of these arsenic species is most likely closely related to the well known oxo-arsenosugars. Due to their symbiotic algae giant clams contain oxo-arsenosugars with Carboxyl-, Carbamate- and Adenine-aglycone in significant amounts. Now we provide evidence for the presence of the thio analogues of these three arsenosugars in tissue extracts of the giant clams Tridacna maxima and Tridacna derasa. Anion-exchange high-performance liquid chromatography (HPLC) online with electrospray tandem mass spectrometry was applied for the identification of the thio-arsenosugars and the indirect quantification via their oxo analogues.

An improved method for isolation and purification of sedimentary porphyrins by high-performance liquid chromatography for compound-specific isotopic analysis

We describe an improved method for purification of sedimentary vanadyl and nickel porphyrins (i.e., naturally occurring metalloalkylporphyrins). For the purpose of compound-specific isotopic analyses, various sedimentary porphyrins were purified from the complex natural mixtures by the dual-step high-performance liquid chromatography (HPLC) method. The high-sample-capacity reversed-phase HPLCs by adding N, N-dimethylformamide to the mobile phase allow an efficient collection of fractions containing the target compounds even using analytical-scale columns. Furthermore, this method achieved improved chromatographic resolutions but significantly reduced the overall retention time down to 60% compared with the previous work. The target compounds were then isolated with the normal-phase HPLC with the baseline-resolution, which is necessary to avoid chromatographic isotopic fractionation. One of the advantages of this method is that it requires neither derivatization nor demetallation. The purity of these isolated compounds was demonstrated by various HPLC online detection methods utilizing a photodiode-array detector, a mass selective detector. The overall recoveries of Ni porphyrin, VO porphyrin, and porphyrin-free base, respectively, were estimated to be approximately 50–60%, 65%, and 85%.

Phototoxicity testing by online irradiation and HPLC

A high-performance liquid chromatography (HPLC) system was developed for the determination of drug photostability and phototoxicity based on an automated column-switching system with aqueous online UV-A irradiation and hyphenated organic separation of the drug and its photoproducts. The photoreactor is built with an poly(ethylene-co-tetrafluoroethylene) (ETFE) reaction coil knitted around a UV-A light source. The chromatographic separation was performed with two special C18 columns, which are also suitable for using with pure water as eluent. Degradation of chlorpromazine (CPZ) by ultraviolet light was investigated at pH 7 and pH 3. Furthermore chlorpromazine was irradiated in the presence of guanosine-5-monophosphate (GMP) in pH 7 buffered solution, leading to a new photoproduct. In the pH 3 irradiation studies of CPZ and GMP, no reaction was detected between the molecules.

Profiling 627 Mitochondrial Nucleotides via the Analysis of a 23-Plex Polymerase Chain Reaction by Liquid Chromatography−Electrospray Ionization Time-of-Flight Mass Spectrometry

We present a rapid and informative mitochondrial DNA profiling system, which has high forensic impact. The assay is based on the analysis of a 23-plex PCR by ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS). In a single 25-min run, an overall number of 627 nucleotide positions were screened. The vast majority of observed sequence variations were explainable by alterations of the allelic states of the 23 target SNPs, which were selected on their ability to increase forensic discrimination within West Eurasian populations. Within an Austrian population sample comprising 90 unrelated men, 14 different, nontarget SNP-related sequence variations--13 base substitutions and 1 deletion--were detected by ICEMS and confirmed by sequencing. All amplified sequences were located outside of the routinely sequenced hypervariable segments (HVS-I and HVS-II) of the noncoding control region. Accordingly, the genetic information obtained by the 23-plex PCR-ICEMS assay could be combined with HVS-I/HVS-II sequencing results to one highly discriminating mtDNA profile, which covered approximately 7.5% of the total mtDNA genome. With the 23-plex PCR-ICEMS assay, DNA mixtures were detected and the allelic ratios were accurately quantified. The observed robustness and sensitivity underlined the practical applicability of the assay in forensic science, which was proven by typing eight representative casework samples.