Reproducible Separation Research Articles

Application of MELC and MEEKC for the Analysis of Paracetamol and Related Impurities in Suppositories

Rapid MELC and MEEKC methods were developed for paracetamol suppository assay. MELC methods for paracetamol analysis and for separation of paracetamol and its impurities were previously reported. In this study, further development of MEEKC methods and a MELC method using anionic and cationic microemulsions gave excellent validation results for paracetamol content in suppositories. SDS Microemulsion instability resulted in poor reproducibility for impurity separations using gradient elution. A novel isocratic CTAB MELC method achieved reproducible separation of paracetamol and its impurities at 0.1% levels. MEEKC methods using SDS and CTAB microemulsions resolved all of the impurities however detection at 0.1% levels was not possible. These methods gave significant benefits in terms of reduced sample pre-treatment requirements. CTAB microemulsions had greater solubilising power than their SDS equivalent and were more stable due to their longer alkyl chain length.

Separation of membrane proteins by two‐dimensional electrophoresis using cationic rehydrated strips

Due to their poor solubility during IEF membrane proteins cannot be separated and analyzed satisfactorily with classical 2-DE. A more efficient method for such hydrophobic proteins is the benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE, but the corresponding protocol is intricate and time-consuming. We now developed an easy-to-handle electrophoresis method in connection with a novel device which enables reproducible separation of ionic solubilized membrane proteins using individually rehydrated plastic sheet gel strips. These strips are suitable for the first dimension in a 2-D 16-BAC/SDS system and can be handled easily; this is demonstrated by the separation of membrane proteins of human embryonic kidney (HEK293) cells.

Development of capillary electrochromatography with poly(styrene-divinylbenzene-vinylbenzenesulfonic acid) monolith as the stationary phase

A new polystyrene-based monolithic stationary phase, which was prepared by single step in situ copolymerization of styrene, divinylbenzene and vinylbenzenesulfonic acid (VBSA), was developed as a separation column for capillary electrochromatography, in which VBSA was employed as the charge-bearing monomer. Polymerization time of the polystyrene-based monolith had slightly influenced the separation time of the tested analytes, but it effectively altered their separation resolutions. Furthermore, baseline separation for a wider range of acetonitrile levels of mobile phase was achieved when a monolithic column prepared by a longer polymerization time was used. This novel polystyrene-based monolithic column provided an adequate electroosmotic flow either in basic or acidic mobile phase when VBSA level was maintained at 2.6% (w/w). Finally, this proposed polystyrene-based column allowed seven tested analytes to achieve a reproducible baseline separation within 2.2 min with theoretical plate numbers higher than 164 000 plates/m.

Comparative LC‐MS: A landscape of peaks and valleys

Quantitative proteomics approaches using stable isotopes are well-known and used in many labs nowadays. More recently, high resolution quantitative approaches are reported that rely on LC-MS quantitation of peptide concentrations by comparing peak intensities between multiple runs obtained by continuous detection in MS mode. Characteristic of these comparative LC-MS procedures is that they do not rely on the use of stable isotopes; therefore the procedure is often referred to as label-free LC-MS. In order to compare at comprehensive scale peak intensity data in multiple LC-MS datasets, dedicated software is required for detection, matching and alignment of peaks. The high accuracy in quantitative determination of peptide abundance provides an impressive level of detail. This approach also requires an experimental set-up where quantitative aspects of protein extraction and reproducible separation conditions need to be well controlled. In this paper we will provide insight in the critical parameters that affect the quality of the results and list an overview of the most recent software packages that are available for this procedure.

Gel-Eluted Liquid Fraction Entrapment Electrophoresis: An Electrophoretic Method for Broad Molecular Weight Range Proteome Separation

Although well-established as a technique for protein purification, the application of continuous elution tube gel electrophoresis to proteome fractionation remains problematic. Difficulties associated with sample collection, particularly at the high mass range or at low sample loadings, continue to plague the technique. Furthermore, an upper mass limit is imposed as slow-moving higher molecular weight proteins are progressively diluted during the collection phase. In short, with current technology, effective separation over a broad mass range has not been achieved. In this work, we present improved techniques for continuous elution tube gel electrophoresis to accommodate broad mass range separation of proteins. Our device enables rapid partitioning of a proteome into discrete mass range fractions in the solution phase. High recovery is achieved at submicrogram to milligram sample loadings. We demonstrate comprehensive, reproducible separations of protein mixtures, as well as separation of a proteome in as fast as 1 h, over mass ranges from below 10 to 250 kDa. Finally, we identified proteins from a prefractionated standard protein mixture using liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis.

On‐line concentration of proteins by SDS‐CGE with LIF detection

We present a simple approach for on-line concentration of SDS-protein complexes by using poly(vinyl alcohol) (PVA) solution in CGE. In comparison to the coated capillary, the presence of EOF in CGE omitted the need to fill the capillaries with polymer solutions prior to the analysis. More importantly, we found that highly reproducible separation of eight proteins by 3.5% PVA was achieved between runs and without the regeneration of high bulk EOF; the RSD of migration times was less than 0.7%. To further improve the concentration sensitivity, neutral PVA was introduced into the capillary with the help of EOF to act as sieving matrix. The occurrence of stacking at the boundary between the PVA and the sample zone is mainly due to the retardation of proteins by PVA. As a result, the LODs at an S/N of 3 for SDS-protein complexes are of the order of sub-nM to several nM. For example, the LOD for BSA is 0.78 nM, which is a 91-fold sensitivity enhancement over the normal injection. In addition, our stacking method has been applied to the analyses of proteins in Escherichia coli cells. The peak for beta-galactosidase (E. coli) was observed after 0.1 microM beta-galactosidase was spiked into the E. coli samples.

Rapid identification of purified enteropathogenic Escherichia coli by microchip electrophoresis

In this work, the potential of PDMS-based microchip electrophoresis in the identifications and characterizations of microorganism was evaluated. Enteropathogenic E. coli (EPEC) was selected as the model microorganism. In this study, separation parameters such as applied voltage, concentrations of buffer and buffer modifier, injection voltage, and duration of injection had been investigated and optimized. Determination of EPEC bacteria could be completed within 2 min with good reproducibility. RSDs were less than 0.5 and 5% in migration time and peak area, respectively. Separation efficiency corresponding to plate number of more than 100,000 was achieved. In order to obtain reproducible separations, sample pretreatment was found to be essential. Microchip electrophoresis with LIF detection could potentially revolutionize certain aspects of microbiology involving diagnosis, profiling of pathogens, environmental analysis, and many other areas of study.

Optimization of oligonucleotide microarray fabricated by spotting 65-mer

DNA microarrays currently provide measurements of sufficiently high quality to allow a wide variety of sound inferences about gene regulation and the coordination of cellular processes to be drawn. Nonetheless, a desire for greater precision in the measurements continues to drive the microarray research community to seek higher measurement quality through improvements in array fabrication and sample labeling and hybridization. We prepared oligonucleotide microarrays by printing 65-mer on aldehyde functional group-derivatized slides as described in a previous study. We could improve the reliability of data by removing enzymatic bias during probe labeling and hybridizing under a more stringent condition. This optimized method was used to profile gene expression patterns for nine different mouse tissues and organs, and multidimensional scaling (MDS) analysis of data showed both strong similarity between like samples and a clear, highly reproducible separation between different tissue samples. Three other microarrays were fabricated on commercial substrates and hybridized following the manufacturer’s instructions. The data were then compared with in-house microarray data and reverse transcription–polymerase chain reaction (RT–PCR) data. The microarray printed on the custom aldehyde slide was superior to microarrays printed on commercially available substrate slides in terms of signal intensities, background, and hybridization characteristics. The data from the custom substrate microarray generally showed good agreement in quantitative changes up to 100-fold changes of transcript abundance with RT–PCR data. However, more accurate comparisons will be made as more genomic sequence information is gathered in the public data domain.

Investigation of high-speed gas chromatography using synchronized dual-valve injection and resistively heated temperature programming

High-speed temperature programming is implemented via the direct resistive heating of the separation column (2.3 m MXT-5 Silicosteel column with a 180 μm I.D. and a 0.4 μm 5% phenyl/95% dimethyl polysiloxane film). Resistive temperature programming was coupled with synchronized dual-valve injection (with an injection pulse width of 2 ms), producing a complete high-speed gas chromatography (GC) system. A comparison of isothermal and temperature programmed separations of seven n-alkanes (C 6 and C 8–C 13) shows a substantial improvement of peak width and peak capacity with temperature programming. The system was further implemented in separations of a mixture of analytes from various chemical classes. Separations of the n-alkane mixture using three different temperature programming rates are reported. A temperature programming rate as high as 240 °C/s is demonstrated. The method for determination of temperature programming rate, based on isothermal data, is discussed. The high-speed resistive column heating temperature programming resulted in highly reproducible separations. The highest rate of temperature programming (240 °C/s) resulted in retention time and peak width RSD, on average, of 0.5 and 1.4%, respectively, for the n-alkane mixture. This high level of precision was achieved with peak widths-at-half-height ranging from 13 to 36 ms, and retention times ranging from 147 to 444 ms (for n-hexane to n-tridecane).

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.

Electrophoretic separations of cerebrospinal fluid proteins in clinical investigations

The cerebrospinal fluid (CSF) is a key sample in the research for novel molecular biomarkers of neurodegenerative disorders. CSF represents a repertoire of neuro-secreted, biosynthesised and metabolised molecular products of the central nervous system (CNS). Diffusion of macromolecules from the peripheral circulatory system to the CSF is highly regulated by the blood-brain barrier, which prevents uncontrolled distribution of proteins in the CNS. The development of reproducible high resolution separations of proteins in 2-D electrophoresis methods by the advent of immobilised pH gradient has opened the route to multivariate holistic protein pattern investigation of CSF into neurodegenerative disorders. Moreover, the introduction of pre-fractionation techniques such as free flow electrophoresis is currently increasing the dynamic depth of proteome analysis. Alzheimer's disease (AD) and other forms of dementia, demyelinating diseases, Parkinson's disease (PD), and Creutzfeldt-Jakob disease (CJD) have been evaluated for biomarker discovery by CSF investigation in multiple studies. However, the statistical design of these clinical cross-sectional investigations remains a limited factor given the strong statistical power required for complex multivariate analysis. These initial evidences are of particular interest in dissecting specific molecular mechanisms. The development of fast and economic profiling of CSF by linear matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) is providing a new ancillary technology to assess sample quality and pre-analytical requirements. In the following we take into account all these issues in the CSF proteomics investigation, especially highlighting the possible application in the development of clinical molecular biomarkers.

Investigation on sources of contamination, for an accurate analytical blank estimation, during measurements of the Fe content in seawater by isotope dilution inductively coupled plasma mass spectrometry

It is well known that direct measurement of the Fe content in seawater by means of inductively coupled plasma mass spectrometry (ICPMS) is hardly possible. For open ocean water samples, typically, it requires a separation from the matrix associated to significant concentration factors. Furthermore, in the low ng kg−1 range, a correct assessment of the analytical blank is of crucial importance. Despite years of experiments and publications, blank estimation remains a fundamental analytical challenge for the realisation of reliable profiles of dissolved Fe data. Not only must this blank be low and reproducible, which is particularly difficult considering the ubiquity of Fe and the complexity of the seawater matrix, but also realistic. Thus, the complete analytical sequence must be applied to real seawater with zero concentration of Fe to produce a “field blank” (FB), according to the International Union of Pure and Applied Chemistry terminology. This paper investigates the sources of contamination and describes two ways of establishing reliable field blank values for the isotope dilution (ID) ICPMS procedure described by Wu and Boyle (J. Wu and E. A. Boyle, Anal. Chim. Acta, 1998, 367, 183), based on a multiple steps protocol, including a co-precipitation with magnesium hydroxide after ammonia loading, and consecutive dissolution with hydrochloric acid. The analytical protocol is optimised to achieve reproducible separation–pre-concentration of ∼100% Fe under stable pH conditions. Typically, at ∼30 ng Fe kg−1 level a concentration factor of up to 15 can be achieved, leading to samples containing residual salinity ∼0.06%. The first approach proposed for field blank determination applies mostly to Fe mass fractions >500 ng kg−1 and results from the comparison of two IDMS-based Fe content values of an identical sample, where the first is produced by Mg(OH)2 co-precipitation and the second after a simple dilution. For the second approach, adapted to lower Fe content samples (down to 3 ng Fe kg−1), the absolute field blank is the intercept of a linear regression between sample masses and corresponding absolute Fe contents for a given set of sample replicates. The estimated field blanks for both approaches were 16 ± 12 ng kg−1 and 6 ± 2 ng kg−1, respectively. We found that manipulations (i.e. sample handling and the environment) are by far the largest source of contamination as they contribute ∼75% of the total. The 2% nitric acid used to dissolve the precipitate and the instrumental background come next, with, respectively, 3–10% and 5–9% contributions. The measurement procedure was validated through the use of reference materials, a systematic assessment of factors influencing the result, by calculating the combined measurement uncertainty, and from the results obtained on a test material of a recent inter-laboratory comparison.

High-Speed MEMS-Based Gas Chromatography

This paper reports microfabricated silicon-glass separation columns for high-speed micro gas chromatography (muGC) systems. The microfabricated columns are integrated with resistive heaters and temperatures sensors and capacitive pressure sensors to allow temperature and pressure programming and flow control and to achieve reproducible separations in a muGC system. These 25-cm-long, 150-mum-wide, and 250-mum-deep columns are fabricated on a 1.2-cm square die using a silicon-on-glass dissolved wafer process. Programmed with temperature ramps of 10 degC/s, the low-mass columns separate eleven-component gaseous mixtures in less than 10 s, including alkanes from C <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">5</sub> to C <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">16</sub> and simulants for C-4, TNT, sarin, and mustard gas. When used in arrayed architectures, these MEMS columns should allow high-speed analysis without sacrificing separation efficiency

Reproducible two-dimensional capillary electrophoresis analysis of Barrett's esophagus tissues.

We have constructed a high-speed, two-dimensional capillary electrophoresis system with a compact and high-sensitivity fluorescence detector. This instrument is used for the rapid and reproducible separations of Barrett's esophagus tissue homogenates. Proteins and biogenic amines are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled biomolecules are separated sequentially in two capillaries. The first capillary employs capillary sieving electrophoresis using a replaceable sieving matrix. Fractions are successively transferred to a second capillary where they undergo additional separation by micellar electrokinetic capillary chromatography. The comprehensive two-dimensional separation requires 60 min. Within-day migration time reproducibility is better than 1% in both dimensions for the 50 most intense features. Between-day migration time precision is 1.3% for CSE and better than 0.6% for MECC. Biopsies were obtained from the squamous epithelium in the proximal tubular esophagus, Barrett's epithelium from the distal esophagus, and fundus region of the stomach from each of three Barrett's esophagus patients with informed consent. We identified 18 features from the homogenate profiles as biogenic amines and amino acids. For each of the patients, Barrett's biopsies had more than 5 times the levels of phenylalanine and alanine as compared to squamous tissues. The patient with high-grade dysplasia shows the highest concentrations for 13 of the amino acids across all tissue types. Concentrations of glycine are 40 times higher in squamous biopsies compared to Barrett's and fundal biopsies from the patient with high-grade dysplasia. These results suggest that two-dimensional capillary electrophoresis may be of value for the rapid characterization of endoscopic and surgical biopsies.

Ultrafast Gas Chromatography on Single-Wall Carbon Nanotube Stationary Phases in Microfabricated Channels

The key to rapid temperature programmed separations with gas chromatography are a fast, low-volume injection and a short microbore separation column with fast resistive heating. One of the major problems with the reduction of column dimensions for micro gas chromatography is the availability of a stationary phase that provides good separation performance. In this report, we present the first integration of single-wall carbon nanotubes (SWNTs) as a stationary phase into 100 mum x 100 mum square and 50-cm-long microfabricated channels. The small size of this column with integrated resistive heater and the robustness of the SWNT phase allow for fast temperature programming of up to 60 degrees C/s. A combination of the fast temperature programming and the narrow peak width of small-volume injections that can be obtained from a high-speed, dual-valve injection system allows for rapid separations of gas mixtures. We demonstrate highly reproducible separations of four-compound test mixtures on these columns in less than 1 s using fast temperature programming.

Determination of carbohydrate-deficient transferrin in human serum using the Bio-Rad %CDT by HPLC test

Background Carbohydrate-deficient transferrin (CDT) in serum is a biomarker used to identify individuals with sustained, heavy alcohol consumption. This study evaluated the performance of a new commercial method for CDT, the Bio-Rad %CDT by HPLC test, that measures relative amounts of separate transferrin glycoforms in proportion to total transferrin. Method The samples used were two human serum pools (low/high disialotransferrin), 150 clinical sera with low to highly elevated disialotransferrin values, and 18 genetic transferrin variants. Glycoforms are separated on a gradient HPLC system, followed by specific measurement of the iron-transferrin complex at 460 nm. Comparison was made with an HPLC candidate reference method on an Agilent 1100 LC system. Results The Bio-Rad %CDT by HPLC test allowed for reproducible separation and quantification of the transferrin glycoforms within ∼ 6 min. Genetic variants were readily identified. For the low and high serum pool, the total CV was 8.5% and 4.3%, respectively. The relative amounts of disialotransferrin, the main CDT glycoform, were in good agreement with the results of the HPLC candidate reference method ( r 2 = 0.998, p < 0.0001). Conclusions The present results demonstrated that the Bio-Rad %CDT by HPLC test is appropriate for confirmatory and routine %CDT testing in human serum, with the added advantage over previously published HPLC methods of an improved serum sample pretreatment and a shorter total analysis time.

Isolation and characterization of human myeloid progenitor populations—TpoR as discriminator between common myeloid and megakaryocyte/erythroid progenitors

The common myeloid progenitor (CMP) and its progeny, the granulocyte/monocyte progenitor (GMP) and megakaryocyte/erythrocyte progenitor (MEP), have been isolated based on the surface expression of CD34, IL-3Ralpha, and CD45RA. However, high resolution of IL-3Ralpha- and IL-3Ralphalo cells required to adequately separate the CMP and MEP populations is difficult to achieve. The aim of this study was to find a complementary surface marker to obtain a better separation of these two populations and to further characterize the acquired progenitor populations. To evaluate the thrombopoietin receptor (TpoR) as a candidate marker, CD19-/CD34+/IL-3Ralphalo/-/CD45RA-/TpoR- (CMP), CD19-/CD34+/IL-3Ralphalo/CD45RA+/TpoR- (GMP), and CD19-/CD34+/IL-3Ralphalo/-/CD45RA-/TpoR+ (MEP) cells from human bone marrow were sorted to semisolid cultures for colony assays, and in addition analyzed for their surface expression of other growth factor receptors (GFRs) and sorted to real-time RT-PCR for gene expression analysis. The colony-formation and gene expression assays showed that inclusion of TpoR as a marker gave a distinct and reproducible separation of the myeloid progenitors. Furthermore, most GFR surface expression correlated to gene expression, but there were also striking discrepancies, in particular for the common beta-chain of the IL-3R, GM-CSFR, and IL-5R and for TpoR. Our data establish the TpoR as an important tool for isolation of the myeloid progenitors and demonstrate that the surface expression of GFRs cannot be predicted by their gene expression. Importantly, the refined isolation of CMPs will allow more detailed studies of regulatory mechanisms steering CMPs towards erythropoiesis vs granulopoiesis in steady state and response to peripheral demands.

Ionene‐dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS. The influence of several CE and MS operating parameters, such as the concentration of CE running buffer, applied external pressure, and the composition and flow rate of sheath liquid on CE-ESI-MS was studied. The results demonstrated that when the capillary was permanently coated with 6,6-ionene and the pH value of acetic acid-ammonium acetate running buffer was 4.80 and 5.50, respectively, a significantly reproducible separation was achieved for rhEPO and uEPO glycoforms. In the online CE-ESI-MS experiments, we not only achieved the online MS signal of uEPO, but also obtained baseline separation of three major rhEPO glycoforms successfully and reproducibly on the 6,6-ionene-coated capillaries. Furthermore, the standard mixture of rhEPO and uEPO was separated, and two incompletely resolved peaks that were identified to be rhEPO and uEPO by the unique MS "fingerprint" were obtained. Additionally, the molecular weight of rhEPO and uEPO were verified and compared to the results by MALDI-TOF-MS. It can be concluded that, in contrast to other indirect methods, the online CE-ESI-MS technique with the combination of the advantages of both CE and MS shows great potential for the separation and detection of rhEPO doping directly in competitive sports.

High-performance temperature-programmed microfabricated gas chromatography columns

This paper reports the first development of high-performance, silicon-glass micro-gas chromatography (/spl mu/GC) columns having integrated heaters and temperature sensors for temperature programming, and integrated pressure sensors for flow control. These 3-m long, 150-/spl mu/m wide and 250-/spl mu/m deep columns, integrated on a 3.3 cm square die, were fabricated using a silicon-on-glass dissolved wafer process. Demonstrating the contributions to heat dissipation from conduction, convection, and radiation with and without packaging, it is shown that using a 7.5-mm high atmospheric pressure package reduces power consumption to about 650 mW at 100/spl deg/C, while vacuum packaging reduces the steady-state power requirements to less than 100 mW. Under vacuum conditions, 600 mW is needed for a temperature-programming rate of 40/spl deg/C/min. The 2300 ppm//spl deg/C TCR of the temperature sensors and the 50 fF/kPa sensitivity of the pressure sensors satisfy the requirements needed to achieve reproducible separations in a /spl mu/GC system. Using these columns, highly resolved 20-component separations were obtained with analysis times that are a factor of two faster than isothermal responses.

Analysis of Biogenic Amine Variability among Individual Fly Heads with Micellar Electrokinetic Capillary Chromatography−Electrochemical Detection

Neurochemical variability among individual Drosophila heads has been examined with the sensitivity of electrochemical detection and the selectivity of micellar electrokinetic capillary chromatography. Homogenization of single Drosophila heads in volumes as small as 100 nL has been accomplished. Here we demonstrate reproducible separations for single fly heads in 250-nL volumes providing a 4-fold increase in sensitivity without overloading the electrochemical detector. This increase in sensitivity allows detection of previously undetected analytes, such as N-acetyltyramine (naTA) and octopamine (OA). Analytes including L-3,4-dihydroxyphenylalanine, N-acetyl octopamine, N-acetyldopamine, naTA, N-acetylserotonin, OA, dopamine, tyramine, and serotonin also have been consistently identified in single-head homogenates and observed with homogenates representing populations of Drosophila. Neurochemical variation between individual flies as well as the consistency within a population indicates varying amounts of neurotransmitter turnover. The inception, design, and fabrication of a miniature tissue homogenizer has enabled the separation of biogenic amines and metabolites from these severely volume-limited single Drosophila head homogenates.