Home-delivered meals for people with dementia: Which model delays nursing home placement? - Protocol for a feasibility pilot.

BACKGROUND: Home-delivered meals promote food security, socialization, and independence among homebound older adults. However, it is unclear which of the two predominant modes of meal delivery, daily-delivered vs. drop-shipped, frozen meals, promotes community living for homebound older adults with dementia. Our objective is to present the protocol for a pilot multisite, two-arm, pragmatic feasibility trial comparing the effect of two modes of meal delivery on nursing home placement among people with dementia. We include justifications for individual randomization with different consent processes and waivers for specific elements of the trial. METHODS: 236 individuals with dementia on waiting lists at three Meals on Wheels programs' in Florida and Texas will be randomized to receive either: 1) meals delivered multiple times per week by a Meals on Wheels volunteer or paid driver who may socialize with and provide an informal wellness check or 2) frozen meals that are mailed to participants' homes every two weeks. We will evaluate and refine processes for recruitment and randomization; assess adherence to the intervention; identify common themes in participant experience; and test processes for linking participant data with Medicare records and nursing home assessment data. We will conduct exploratory analyses examining time to nursing home placement, the primary outcome for the larger trial. CONCLUSION: This pilot will inform the follow-on large-scale, definitive pragmatic trial. In addition, the justifications for individual randomization with differing consent procedures for elements of a pragmatic trial provide a model for future trialists looking to develop ethical and feasible pragmatic studies enrolling people with dementia.

Phase 1 Clinical Trial of a Conditionally Replication-Defective Human Cytomegalovirus (CMV) Vaccine in CMV-Seronegative Subjects.

BACKGROUND: A conditionally replication-defective human cytomegalovirus (CMV) vaccine (V160) derived from AD169 and genetically engineered to express CMV pentameric complex (gH/gL/pUL128/pUL130/pUL131) was developed and evaluated for phase 1 vaccine safety and immunogenicity in CMV-seronegative and CMV-seropositive adults. METHODS: Subjects received 3 doses of V160 or placebo on day 1, month 1, and month 6. Four vaccine dose levels, formulated with or without aluminum phosphate adjuvant, were evaluated. Injection-site and systemic adverse events (AEs) and vaccine viral shedding were monitored. CMV-specific cellular and humoral responses were measured by interferon-gamma ELISPOT and virus neutralization assay up to 12 months after last dose. RESULTS: V160 was generally well-tolerated, with no serious AEs observed. Transient, mild-to-moderate injection-site and systemic AEs were reported more frequently in vaccinated subjects than placebo. Vaccine viral shedding was not detected in any subject, confirming the nonreplicating feature of V160. Robust neutralizing antibody titers were elicited and maintained through 12 months postvaccination. Cellular responses to structural and nonstructural viral proteins were observed, indicating de novo expression of viral genes postvaccination. CONCLUSIONS: V160 displayed an acceptable safety profile. Levels of neutralizing antibodies and T-cell responses in CMV-seronegative subjects were within ranges observed following natural CMV infection. CLINICAL TRIAL REGISTRATION: . NCT01986010.

Biochemical characterization of an isoprene synthase from Campylopus introflexus (heath star moss).

Each year, plants emit terragram quantities of the reactive hydrocarbon isoprene (2-methyl-1,3-butadiene) into the earth's atmosphere. In isoprene-emitting plants, the enzyme isoprene synthase (ISPS) catalyzes the production of isoprene from the isoprenoid intermediate dimethylallyl diphosphate (DMADP). While isoprene is emitted from all major classes of land plants, to date ISPSs from angiosperms only have been characterized. Here, we report the identification and initial biochemical characterization of a DMADP-dependent ISPS from the isoprene-emitting bryophyte Campylopus introflexus (heath star moss). The partially-purified C. introflexus ISPS (CiISPS) exhibited a Km for DMADP of 0.37 ± 0.28 mM, a pH optimum of 8.6 ± 0.5, and a temperature optimum of 40 ± 3 °C in vitro. Like ISPSs from angiosperms, the CiISPS required the presence of a divalent cation. However, unlike angiosperm ISPSs, the CiISPS utilized Mn(2+) preferentially over Mg(2+). Efforts are currently underway in our laboratory to further purify the CiISPS and clone the cDNA sequence encoding this novel enzyme. Our discovery of the first bryophyte ISPS paves the way for future studies concerning the evolutionary origins of isoprene emission in land plants and may help generate new bryophyte model systems for physiological and biochemical research on plant isoprene function.

The effect of cervical spine manipulation on postural sway in patients with nonspecific neck pain.

OBJECTIVE: This crossover study aimed to determine whether a single high-velocity, low-amplitude manipulation of the cervical spine would affect postural sway in adults with nonspecific neck pain. METHODS: Ten participants received, in random order, 7 days apart, a high-velocity, low-amplitude manipulation applied to a dysfunctional spinal segment and a passive head-movement control. Four parameters of postural sway were measured before, immediately after, and at 5 and 10 minutes after each procedure. RESULTS: Results showed no differences between interventions in change in any of the parameters. When changes before and immediately after each procedure were analyzed separately, only the control showed a significant change in the length of center of pressure path (an increase from median, 118 mm; interquartlie range, 93-137 mm to an increase to 132 mm; 112-147; P = .02). CONCLUSION: This study failed to show evidence that single manipulation of the cervical spine influenced postural sway. Given the ability of the postural control system to reweight the hierarchy of sensory information to compensate for inadequacies in any 1 component, it is possible that any improvements in the mechanisms controlling postural sway elicited by the manipulative intervention may have been concealed.

Environmental regulation of plant gene expression: an RT-qPCR laboratory project for an upper-level undergraduate biochemistry or molecular biology course.

We present a novel laboratory project employing "real-time" RT-qPCR to measure the effect of environment on the expression of the FLOWERING LOCUS C gene, a key regulator of floral timing in Arabidopsis thaliana plants. The project requires four 3-hr laboratory sessions and is aimed at upper-level undergraduate students in biochemistry or molecular biology courses. The project provides students with hands-on experience with RT-qPCR, the current "gold standard" for gene expression analysis, including detailed data analysis using the common 2-ΔΔCT method. Moreover, it provides a convenient starting point for many inquiry-driven projects addressing diverse questions concerning ecological biochemistry, naturally occurring genetic variation, developmental biology, and the regulation of gene expression in nature.

GABA(A) alpha-1 subunit mediated desynchronization of elevated low frequency oscillations alleviates specific dysfunction in stroke--a case report.

OBJECTIVE: The paradoxical effects of the hypnotic imidazopyridine zolpidem, widely reported in persistent vegetative state, have been replicated recently in brain-injured and cognitively impaired patients. However, the neuronal mechanisms underlying these benefits are yet to be demonstrated. We implemented contemporary neuroimaging methods to investigate sensorimotor and cognitive improvements, observed in stroke patient JP following zolpidem administration. METHODS: We used Magnetic-Resonance-Imaging (MRI) and Magnetic-Resonance-Spectroscopy (MRS) to anatomically and chemically characterize stroke damage. Single-photon-emission-computed-tomography (SPECT) and magnetoencephalography (MEG) were used to identify changes in cerebrovascular perfusion and neuronal network activity in response to sub-sedative doses of zolpidem, zopiclone and placebo. Cognitive improvements were measured using the WAIS-III and auditory-verbal tasks. RESULTS: MRI and MRS revealed a lesion with complete loss of neuronal viability in the left temporal-parietal region; whilst SPECT indicated improved perfusion in the affected hemisphere following zolpidem. MEG demonstrated high-amplitude theta (4-10 Hz) and beta (15-30 Hz) oscillations within the peri-infarct region, which reduced in power coincident with zolpidem uptake and improvements in cognitive and motor function. CONCLUSIONS: In JP, functional deficits and pathological oscillations appear coincidentally reduced following administration of low-dose zolpidem. SIGNIFICANCE: GABA(A) alpha-1 sensitive desynchronisation of pathological oscillations may represent a biomarker and potential therapeutic target in brain injury.

Establishment of the Yellow Starthistle Rust in California: Release, Recovery, and Spread.

The rust fungus Puccinia jaceae var. solstitialis is the first pathogen released for biological control of yellow starthistle (Centaurea solstitialis). From 2004 to 2006, the pathogen was released at 176 sites in 40 counties throughout the state of California. Release sites were evaluated 1 to 3 months and 1, 2, and, in some cases, 3 years after releases to monitor for reemergence. At 1 to 3 months after inoculation, 58 to 93% of sites had rust infection, depending on the year. After 1, 2, and 3 years, the percentages declined to 19 to 21, 9 to 10, and 3% respectively. Spread was detected at 19% of the sites with rust infection, with an average distance of 21 m (±13.3 standard error). The greatest spread occurred at a site in Sonoma County. At this site, the rust spread to over 37 acres 1 year after it was released and has remained in this area for three seasons. Reemergence 1 and 2 years after inoculations was more likely in Northern (above 40°N) compared with Southern California (below 36°N). In general, reemergence was more likely at lower elevations when release sites were within 150 km of the coast. Overall, the rust has not demonstrated a strong record of persistence based on these observations.

Interhemispheric differences of spectral power in expressive language: a MEG study with clinical applications.

In the last decade we have seen an exponential growth of functional imaging studies investigating multiple aspects of language processing. These studies have sparked an interest in applying some of the paradigms to various clinically relevant questions, such as the identification of the cortical regions mediating language function in surgical candidates for refractory epilepsy. Here we present data from a group of adult control participants in order to investigate the potential of using frequency specific spectral power changes in MEG activation patterns to establish lateralisation of language function using expressive language tasks. In addition, we report on a paediatric patient whose language function was assessed before and after a left hemisphere amygdalo-hippocampectomy. Our verb generation task produced left hemisphere decreases in beta-band power accompanied by right hemisphere increases in low beta-band power in the majority of the control group, a previously unreported phenomenon. This pattern of spectral power was also found in the patient's post-surgery data, though not her pre-surgery data. Comparison of pre and post-operative results also provided some evidence of reorganisation in language related cortex both inter- and intra-hemispherically following surgery. The differences were not limited to changes in localisation of language specific cortex but also changes in the spectral and temporal profile of frontal brain regions during verb generation. While further investigation is required to establish concordance with invasive measures, our data suggest that the methods described may serve as a reliable lateralisation marker for clinical assessment. Furthermore, our findings highlight the potential utility of MEG for the investigation of cortical language functioning in both healthy development and pathology.

Determination of sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry.

High turbulence liquid chromatography (HTLC, or turbulent flow online extraction) and tandem mass spectrometry (MS/MS) methods for the determination of sitagliptin in human urine and hemodialysate were developed and validated to support clinical studies. A narrow bore large particle size reversed-phase column (Cyclone, 50 mm x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 mm x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. For the urine assay, the LLOQ was 0.1 microg/ml, the linear calibration range was 0.1 to 50 microg/ml, the interday precision (R.S.D.%, n=5) was 2.3-6.5%, and the accuracy was 96.9-106% of the nominal value. For the urine quality control samples (QCs), the intraday precision (R.S.D.%, n=5) and accuracy were 1.8-2.6% and 96.2-106% of the nominal value, respectively. The interday precision (R.S.D.%) for 56 sets of urine QCs over a 6-month period varied from 3.8% to 5.5% and the accuracy from 102% to 105% of the nominal value. For the hemodialysate assay, the LLOQ was 0.01 ng/ml, the linear dynamic range was 0.01-5.0 ng/ml, the interday precision was 1.6-4.1%, and the accuracy was 89.8-104% of the nominal value. For hemodialysate QCs, the intraday precision and accuracy varied from 2.3% to 8.9% and from 99.8% to 111% of the nominal value, respectively. These results demonstrated that both methods are selective, accurate, precise, reproducible, and suitable for quantifying sitagliptin in hemodialysate and human urine samples.

Pharmacokinetics of ertapenem in patients with varying degrees of renal insufficiency and in patients on hemodialysis.

Ertapenem is a parenteral beta-lactam carbapenem antibiotic. This open-label study examined the pharmacokinetics of single 1-g intravenous doses of ertapenem, administered over 30 minutes, in patients with mild, moderate, and advanced renal insufficiency (RI) and in patients with end-stage renal disease (ESRD) requiring hemodialysis. Pharmacokinetics were compared with historical controls pooled across healthy young and elderly subjects. Area under the concentration-time curve from time zero to infinity increased 7% in mild, 53% in moderate, 158% in advanced RI, and 192% in ESRD; end of infusion concentration changed minimally; half-life was 4.5 hours in the historical control group and 4.4, 6.1, 10.6, and 14.1 hours in mild RI, moderate RI, advanced RI, and ESRD, respectively. Hemodialysis cleared approximately 30% of the dose. The recommended dose in mild to moderate RI and after hemodialysis is unchanged at 1 g daily; and in advanced RI and ESRD is reduced to 0.5 g daily. If the daily dose is given 6 hours prior to hemodialysis, a supplementary 150-mg dose (30% of the daily dose) is recommended postdialysis.

Determination of MK-0431 in human plasma using high turbulence liquid chromatography online extraction and tandem mass spectrometry.

A robust and sensitive method using high turbulence liquid chromatography (HTLC) online extraction with tandem mass spectrometry (MS/MS) for the determination of MK-0431 in human plasma was developed and validated to support the clinical studies. This HTLC online extraction method eliminated the time-consuming offline sample extraction procedures and significantly increased productivity. A narrow bore large particle size reversed-phase column (Cyclone, 50 x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. The linear dynamic range of the calibration curve was 0.5 to 1000 ng/mL. Intraday validation was conducted using five calibration curves prepared in five lots of human control plasma, and the intraday precision (RSD%) was from 2.4 to 9.0% and the accuracy was from 98.0 to 103% of the nominal value. The intraday precision (RSD%, n = 5) for plasma quality control (QC) samples varied from 2.0 to 5.3% and accuracy from 103 to 105% of the nominal value. The interday precision (RSD%) for 100 sets of plasma QC samples in 29 analytical runs varied from 6.3 to 9.0% and the accuracy from 98.8 to 104% of the nominal value. No significant difference was observed between the interday and intraday precision and accuracy of the QC samples.

Abnormality of mismatch negativity in response to tone omission in dyslexic adults.

Evidence of abnormalities in the perception of rapidly presented sounds in dyslexia has been interpreted as evidence of a prolonged time window within which sounds can influence the perception of temporally surrounding sounds. We recorded the magnetic mismatch negativity (MMNm) to infrequent tone omissions in a group of six dyslexic adults and six IQ and age-matched controls. An MMNm is only elicited in response to a complete stimulus omission when successive inputs fall within the temporal window of integration (stimulus onset asynchrony (SOA) approximately 160 ms). No MMNm responses were recorded in either experimental group when stimuli were presented at SOAs falling just outside the temporal window of integration (SOA = 175 ms). However, while presentation rates of 100 ms resulted in MMNm responses for all control participants, the same stimulus omissions elicited an MMNm response in only one of the six dyslexic participants. These results cannot support the hypothesis of a prolonged time window of integration, but rather indicate auditory grouping deficits in the dyslexic population.

A new approach for evaluating carryover and its influence on quantitation in high-performance liquid chromatography and tandem mass spectrometry assay.

In a high-performance liquid chromatography (HPLC)-based analytical method, carryover denotes one type of systematic error that is derived from a preceding sample and introduced into the next sample. For typical bioanalytical method development, a significant amount of time and resources are spent on reducing carryover for some analytes. In this paper, the statistical characteristics of carryover were analyzed based on the experimental results. The relative carryover (RC), defined as the peak area ratio of a blank sample to the preceding sample, was constant for the analyte and independent of the concentration of the preceding sample. The influence of carryover on the quantitation of the next injected sample or the 'following' sample was proportional to the concentration ratio of two consecutive samples and the relative carryover. Based on these experiments and analyses, the influence of carryover on the quantitation of unknown samples in an HPLC assay can be evaluated by the estimated carryover influence (ECI), which is the product of the relative carryover and the concentration ratio. This new approach provides a quantitative estimation for the influence of carryover on the quantitation of the unknown sample, and removes the limit put on the dynamic range of the assay by the current criterion of carryover. In general, if the relative standard deviation (RSD) of a validated bioanalytical method is less than 10%, the carryover will not have a significant effect on the accuracy of the assay when the estimated carryover influence is less than 5%.

Geographic distribution and diversity in Claviceps purpurea from salt marsh habitats and characterization of Pacific coast populations.

Claviceps purpurea specific to grasses in salt marsh habitats (Group G3) has previously been identified on Spartina spp. in two locations: New Jersey, USA and southern England. We have identified this subgroup of C. purpurea (G3) in 11 distinct populations including western Europe, South America, and along the Atlantic and Pacific Coasts of the USA. In addition, G3 C. purpurea was discovered on a new host grass genus, Distichlis. Unweighted pair group mean analyses of AFLP and RAPD data reveal distinct structure in G3 C. purpurea populations. Pacific coast populations show little diversity, suggesting they may have been introduced recently in that region. 43 isolates, representing 11 populations were identified as G3 based on the presence of an EcoRI restriction site in the 5.8S ribosomal DNA, and a clear genetic separation from isolates representing the other two C. purpurea subgroups (G1 and G2). In addition, all isolates originating from Spartina densiflora, S. foliosa, S. alterniflora, and S. anglica were identified as belonging to G3. RAPD and AFLP analyses supported the recognition of three discrete groups within C. purpurea and revealed high genetic variability between groups, with only 1.8% of polymorphic markers shared across all isolates. Similarly, analysis of molecular variation (AMOVA) revealed that genetic variability was mainly due to variations between groups (63.5%) rather than within groups (28.5%) or within populations (7.96%). G3 isolates were 35% similar, Pacific coast isolates 83% similar. Ninety percent similarity among isolates from the San Francisco Bay Area suggests this is a recently introduced population.

High-throughput liquid chromatography for drug analysis in biological fluids: investigation of extraction column life.

A novel method was developed and assessed to extend the lifetime of extraction columns of high-throughput liquid chromatography (HTLC) for bioanalysis of human plasma samples. In this method, a 15% acetic acid solution and 90% THF were respectively used as mobile phases to clean up the proteins in human plasma samples and residual lipids from the extraction and analytical columns. The 15% acetic acid solution weakens the interactions between proteins and the stationary phase of the extraction column and increases the protein solubility in the mobile phase. The 90% THF mobile phase prevents the accumulation of lipids and thus reduces the potential damage on the columns. Using this novel method, the extraction column lifetime has been extended to about 2000 direct plasma injections, and this is the first time that high concentration acetic acid and THF are used in HTLC for on-line cleanup and extraction column lifetime extension.

Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection.

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 microg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (> or = 150 microl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (< 0.05 microg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025-10 microg/ml) and quality control samples (0.1, 0.5, and 2.5 microg/ml) were < 6.2% coefficient of variation and 96.8-104.0% of nominal concentration, respectively.

A direct injection high-throughput liquid chromatography tandem mass spectrometry method for the determination of a new orally active alphavbeta3 antagonist in human urine and dialysate.

A generic high-throughput liquid chromatography (HTLC) tandem mass spectrometry (MS/MS) assay for the determination of compound I in human urine and dialysate (hemodialysis) was developed and validated. By using the HTLC on-line extraction technique, sample pretreatment was not necessary. The sample was directly injected onto a narrow bore large particle size extraction column (50 x 1.0 mm, 60 microm) where the sample matrix was rapidly washed away using a high flow rate (5 mL/min) aqueous mobile phase while analytes were retained. The analytes were subsequently eluted from the extraction column onto an analytical column using an organic-enriched mobile phase prior to mass spectrometric detection. The analytes were then eluted from the analytical column to the mass spectrometer for the determination. The linear dynamic range was 2.0-6000 ng/mL for the urine assay and 0.1-300 ng/mL for the dialysate assay. Intraday accuracy and precision were evaluated by analyzing five replicates of calibration standards at all concentrations used to construct the standard curve. For the urine assay, the precision (RSD%, n=5) ranged from 1.9 to 8.0% and the accuracy ranged from 87.8 to 105.2% of nominal value. For the dialysate assay, the precision (RSD%, n=5) ranged from 1.1 to 10.0% and the accuracy from 94.5 to 105.2% of nominal value. In-source fragmentation of the acyl glucuronide metabolite (compound III) did not interfere with the determination of parent compound I. The developed HTLC/MS/MS methodology was specific for compound I in the presence of compound III. Column life-time is increased and sample analysis time is decreased over traditional reversed-phase methods when direct injection assays for urine and dialysate are coupled with the technology of HTLC.

Pharmacokinetics of intramuscularly administered ertapenem.

Ertapenem (INVANZ) is a new once-a-day parental beta-lactam antimicrobial agent that has been shown to be highly effective as a single agent for treatment of various community-acquired and mixed infections. The plasma pharmacokinetics of a 1-g intramuscular (i.m.) dose was compared with those of a 1-g intravenous (i.v.) dose infused over 30 min, the recommended rate of i.v. infusion for comparison, and over 120 min, which more closely mimicked the time course for absorption of the i.m. form. In a three-period crossover study (Part A), 26 healthy subjects received single doses of ertapenem administered i.m., i.v. infused over 30 min, and i.v. infused over 120 min. Blood for ertapenem analysis was collected over 24 h postdose for each treatment. In Part B, these fasted subjects received a 1-g i.m. dose of ertapenem once daily for 7 days. Following a 1-g i.m. dose and a 1-g i.v. dose infused over 120 min, the geometric mean area under the concentration curve from hour 0 to infinity (AUC(0- infinity )) was 541.8 micro g. hr/ml following i.m. administration and 591.4 micro g. hr/ml following a 120-min infusion; the geometric mean ratio was 0.92 with a 90% confidence interval of 0.88 to 0.95. The geometric mean AUC(0- infinity ) was nearly identical when 1-g doses were infused over 30 or 120 min. Although the maximum concentration of drug in serum was somewhat lower following i.m. administration than following i.v. administration, the shape of the plasma concentration profiles was roughly comparable at later time points. Ertapenem did not accumulate after multiple 1-g i.m. daily doses over 7 days. The geometric mean ratio for AUC(0-24) (day 7/day 1) was 0.98 with a 90% confidence interval of 0.94 to 1.02. Thus, the relative bioavailability of the 1-g i.m. dose was 92%. Ertapenem does not accumulate following multiple daily 1-g i.m. doses over 7 days.

A semi-automated 96-well protein precipitation method for the determination of montelukast in human plasma using high performance liquid chromatography/fluorescence detection.

A simple, semi-automated, protein precipitation assay for the determination of montelukast (SINGULAIR, MK-0476) in human plasma has been developed. Montelukast is a potent and selective antagonist of the cysteinyl leukotriene receptor used for the treatment of asthma. A Packard MultiPROBE II EX is used to transfer 300 microl of plasma from sample, standard, and QC sample tubes to a microtiter plate (96-well). After addition of the internal standard by a repeating pipettor, a Tomtec QUADRA 96 adds 400 microl of acetonitrile to all plasma sample wells, simultaneously, in the microtiter plate. The Tomtec is also used to transfer the acetonitrile supernatant from the plasma protein precipitation step, batchwise, to another microtiter plate for analysis by HPLC with fluorescence detection. This assay has been validated and implemented for a clinical study of over 1300 plasma samples and is comparable to manual assays in the LLOQ (lower limit of quantitation, 3 ng/ml) and in stability. This is the first semi-automated protein precipitation assay published for the analysis of montelukast in human plasma and it results in significant time savings over the manual methods, both in sample preparation and in HPLC run time.