Quantitative surface-enhanced Raman spectroscopy of single bases in oligodeoxynucleotides.

To address the question of whether the SERS signals of ss-DNA are simply combinations of the signals from the individual bases that comprise the sequence, SERS spectra of unmodified ss-DNA sequences were obtained using a hydroxylamine-reduced Ag colloid aggregated with MgSO4. Initially, synthetic oligodeoxynucleotides with systematic structural variations were used to investigate the effect of adding single nucleobases to the 3' terminus of 10-mer and 20-mer sequences. It was found that the resulting SERS difference spectra could be used to identify the added nucleobases since they closely matched reference spectra of the same nucleobase. Investigation of the variation in intensity of an adenine probe which was moved along a test sequence showed there was a small end effect where nucleobases near the 3' terminus gave slightly larger signals but the effect was minor (30%). More significantly, in a sample set comprising 25-mer sequences where A, T or G nucleobases were substituted either near the centres of the sequences or the 5' or 3' ends, the SERS difference spectra only matched the expected form in approximately half the cases tested. This variation appeared to be due to changes in secondary structure induced by altering the sequences since uncoiling the sequences in a thermal pre-treatment step gave difference spectra which in all cases matched the expected form. Multivariate analysis of the set of substitution data showed that 99% of the variance could be accounted for in a model with just three factors whose loadings matched the spectra of the A, T, and G nucleobases and which contained no positional information. This suggests that aside from the differences in secondary structure which can be eliminated by thermal pre-treatment, the SERS spectra of the 25-mers studied here are simply the sum of their component parts. Although this means that SERS provides very little information on the primary sequence it should be excellent for the detection of post-transcription modifications to DNA which can occur at multiple positions along a given sequence.

Infrared and Raman screening of seized novel psychoactive substances: a large scale study of >200 samples.

The potential of IR absorption and Raman spectroscopy for rapid identification of novel psychoactive substances (NPS) has been tested using a set of 221 unsorted seized samples suspected of containing NPS. Both IR and Raman spectra showed large variation between the different sub-classifications of NPS and smaller, but still distinguishable, differences between closely related compounds within the same class. In initial tests, screening the samples using spectral searching against a limited reference library allowed only 41% of the samples to be fully identified. The limiting factor in the identification was the large number of active compounds in the seized samples for which no reference vibrational data were available in the libraries rather than poor spectral quality. Therefore, when 33 of these compounds were independently identified by NMR and mass spectrometry and their spectra used to extend the libraries, the percentage of samples identified by IR and Raman screening alone increased to 76%, with only 7% of samples having no identifiable constituents. This study, which is the largest of its type ever carried out, therefore demonstrates that this approach of detecting non-matching samples and then identifying them using standard analytical methods has considerable potential in NPS screening since it allows rapid identification of the constituents of the majority of street quality samples. Only one complete feedback cycle was carried out in this study but there is clearly the potential to carry out continuous identification/updating when this system is used in operational settings.

Surface-enhanced Raman spectroscopy of novel psychoactive substances using polymer-stabilized Ag nanoparticle aggregates.

A set of seized "legal high" samples and pure novel psychoactive substances have been examined by surface-enhanced Raman spectroscopy using polymer-stabilized Ag nanoparticle (Poly-SERS) films. The films both quenched fluorescence in bulk samples and allowed identification of µg quantities of drugs collected with wet swabs from contaminated surfaces.

Isolation and structural determination of non-racemic tertiary cathinone derivatives.

The racemic tertiary cathinones N,N-dimethylcathinone (1), N,N-diethylcathinone (2) and 2-(1-pyrrolidinyl)-propiophenone (3) have been prepared in reasonable yield and characterized using NMR and mass spectroscopy. HPLC indicates that these compounds are isolated as the anticipated racemic mixture. These can then be co-crystallized with (+)-O,O'-di-p-toluoyl-D-tartaric, (+)-O,O'-dibenzoyl-D-tartaric and (−)-O,O'-dibenzoyl-L-tartaric acids giving the single enantiomers S and R respectively of 1, 2 and 3, in the presence of sodium hydroxide through a dynamic kinetic resolution. X-ray structural determination confirmed the enantioselectivity. The free amines could be obtained following basification and extraction. In methanol these are reasonably stable for the period of several hours, and their identity was confirmed by HPLC and CD spectroscopy.

Simple preparation of positively charged silver nanoparticles for detection of anions by surface-enhanced Raman spectroscopy.

Modification of citrate and hydroxylamine reduced Ag colloids with thiocholine bromide, a thiol functionalized quaternary ammonium salt, creates particles where the zeta potential is switched from the normal values of ca.-50 mV to ca. +50 mV. These colloids are stable but can be aggregated with metal salts in much the same way as the parent colloids. They are excellent SERS substrates for detection of anionic targets since their positive zeta potentials promote adsorption of negatively charged ions. This is important because the vast majority of published SERS studies involve cationic or neutral targets. Moreover, the fact that the modifier is a quaternary ammonium ion means that the negative surface charge is maintained even at alkaline pH. The modified colloids can be used to detect compounds which cannot be detected using conventional negatively-charged citrate or hydroxylamine reduced metal nanoparticles, for example the detection limit was 5.0 × 10(-5) M for perchlorate and <8.7 × 10(-7) M for tetraphenylporphine tetrasulfonic acid (TPPS). More importantly, picric acid (an explosive) and diclofenac (a non-steroidal anti-inflammatory) could also be analysed quantitatively at low concentrations, 2.5 × 10(-5) M and 1.9 × 10(-5) M, respectively. Interestingly, the correct choice of aggregating agent is important for achieving high sensitivity since the anion in the aggregating salt may compete with anionic targets for surface binding sites. Finally, since the modification procedure simply involves reaction of nanoparticles with a small alkyl thiol derivative, it can easily be adapted to other particle morphologies or metals.

Propofol-remifentanil TCI sedation for oral surgery.

BACKGROUND AND OBJECTIVES: To investigate the safety and efficacy of a propofol-remifentanil target controlled infusion (TCI) sedation technique for oral surgery. METHODS: This prospective study involved patient and registered nurse (RN) questionnaires to assess the efficacy of sedation during surgery. Outpatients having dental extractions in a private office-based practice with TCI sedation were monitored with a Sleep Apnoea Monitor (SAM) to measure the number of Oxygen Desaturation Events (ODEs), defined as a drop in blood oxygenation to below 94%. RESULTS: Patient- and RN-assessed questionnaires showed a high standard of perceived sedation efficacy, independent of patient BMI. The proportion of patients having one or more ODEs was greater in higher BMI categories: underweight (20.0%), normal (47.9%), overweight (68.2%) and obese (81.8%). The odds of at least one ODE was estimated to be 1.2 times greater for each unit increase in BMI (OR 1.2; 95% CI 1.1-1.3), and at a fixed BMI, the odds of at least one ODE was estimated to be 2.6 times as great for a male as a female (OR 2.6; 95% CI 1.2-5.52). Age, patient nervousness and American Society of Anesthesiologists (ASA) classification were not associated with ODEs. CONCLUSIONS: The TCI technique had a high standard of efficacy, and there were no adverse safety outcomes. Higher BMI and male sex were found to be independently associated with predictors of ODEs during oral surgery under propofol-remifentanil TCI sedation.

Ultraviolet Resonance Raman spectroscopy used to study formulations of salmon calcitonin, a starch-peptide conjugate and TGF-ß3.

Ultraviolet Resonance Raman (UVRR) spectroscopy with excitation at 244 nm was investigated here as a possible useful tool for fast characterization of biopharmaceuticals. Studies were performed on three protein drugs: salmon calcitonin (sCT), starch-peptide conjugate, and transforming growth factor-ß3 (TGF-ß3) adsorbed onto solid granules of tricalcium phosphate (TCP). Secondary structure of sCT was investigated for solutions of 0.5mg/mL up to 200mg/mL, regardless of the turbidity or aggregation states. An increase in ß-sheet content was detected when sCT solutions aggregated. UVRR spectroscopy also detected a small amount of residual organic solvent in a starch-peptide conjugate solution containing only 40 µg/mL of peptide. UVRR spectroscopy was then used to characterize a protein, TGF-ß3, adsorbed onto solid granules of TCP at 50 and 250 µg/cm(3). This study shows that UVRR is suitable to characterize the protein formulations in a broad range of concentrations, in liquid, aggregated, and solid states.

Determination of hydrogen peroxide concentration using a handheld Raman spectrometer: Detection of an explosives precursor.

It has been shown that a handheld Raman spectrometer can be used to determine hydrogen peroxide concentration in aqueous solutions in seconds. To allow quantitative analysis, the aqueous peroxide samples were mixed 50/50 (v/v) with a 4mol/dm(3) sodium perchlorate solution which acted as the internal standard. Standard calibration using relative peak heights of the strongest perchlorate (932cm(-1)) and peroxide bands (876cm(-1)) gave an average error of 1.43% for samples in the range 5-30% peroxide. PLS regression of the same data set gave an average error of 0.98%. In addition, the concentrations of the samples were estimated by searching spectra against a library of standard spectra prepared using the same range of peroxide concentrations at 5% increments and with the same perchlorate internal standard. It was found that the library searching method classified all the test samples correctly, matching either the spectra of the same concentration, if they were present, or matching to the closest concentration if an exact match was not possible. This method thus provides a very rapid technique to allow determination of hydrogen peroxide concentrations in the field, for example at suspected improvised explosives manufacturing sites, without complex calibration procedures.

A novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial.

The association of genetic lesions detected by fluorescence in situ hybridization (FISH) with survival was analyzed in 1069 patients with newly presenting myeloma treated in the Medical Research Council Myeloma IX trial, with the aim of identifying patients associated with the worst prognosis. A comprehensive FISH panel was performed, and the lesions associated with short progression-free survival and overall survival (OS) in multivariate analysis were +1q21, del(17p13) and an adverse immunoglobulin heavy chain gene (IGH) translocation group incorporating t(4;14), t(14;16) and t(14;20). These lesions frequently co-segregated, and there was an association between the accumulation of these adverse FISH lesions and a progressive impairment of survival. This observation was used to define a series of risk groups based on number of adverse lesions. Taking this approach, we defined a favorable risk group by the absence of adverse genetic lesions, an intermediate group with one adverse lesion and a high-risk group defined by the co-segregation of >1 adverse lesion. This genetic grouping was independent of the International Staging System (ISS) and so was integrated with the ISS to identify an ultra-high-risk group defined by ISS II or III and >1 adverse lesion. This group constituted 13.8% of patients and was associated with a median OS of 19.4 months.

Combined antenna and localized plasmon resonance in Raman scattering from random arrays of silver-coated, vertically aligned multiwalled carbon nanotubes.

The electric field enhancement associated with detailed structure within novel optical antenna nanostructures is modeled using the surface integral equation technique in the context of surface-enhanced Raman scattering (SERS). The antennae comprise random arrays of vertically aligned, multiwalled carbon nanotubes dressed with highly granular Ag. Different types of "hot-spot" underpinning the SERS are identified, but contrasting characteristics are revealed. Those at the outer edges of the Ag grains are antenna driven with field enhancement amplified in antenna antinodes while intergrain hotspots are largely independent of antenna activity. Hot-spots between the tops of antennae leaning towards each other also appear to benefit from antenna amplification.

Recent applications of Chemical Imaging to pharmaceutical process monitoring and quality control.

Chemical Imaging (CI) is an emerging platform technology that integrates conventional imaging and spectroscopy to attain both spatial and spectral information from an object. Vibrational spectroscopic methods, such as Near Infrared (NIR) and Raman spectroscopy, combined with imaging are particularly useful for analysis of biological/pharmaceutical forms. The rapid, non-destructive and non-invasive features of CI mark its potential suitability as a process analytical tool for the pharmaceutical industry, for both process monitoring and quality control in the many stages of drug production. This paper provides an overview of CI principles, instrumentation and analysis. Recent applications of Raman and NIR-CI to pharmaceutical quality and process control are presented; challenges facing CI implementation and likely future developments in the technology are also discussed.

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.

Differential gene expression during capillary morphogenesis in 3D collagen matrices: regulated expression of genes involved in basement membrane matrix assembly, cell cycle progression, cellular differentiation and G-protein signaling.

We have performed a screening analysis of differential gene expression using a defined in vitro model of human capillary tube formation. Gene array, differential display and cDNA library screening were used to identify both known and novel differentially expressed genes. Major findings include: the upregulation and functional importance of genes associated with basement membrane matrix assembly; the upregulation of growth factors, transcription factors, anti-apoptotic factors, markers of endothelial cell differentiation, JAK-STAT signalling molecules, adhesion receptors, proteinase inhibitors and actin regulatory proteins; and expression changes consistent with inhibition of cell cycle progression, increased cholesterol biosynthesis, decreased ubiquitin-proteasome mediated degradation, and activation of G-protein signaling pathways. Using DNA microarray analysis, the most induced genes at 8, 24 and 48 hours compared with those at 0 hours were jagged-1, stanniocalcin and angiopoietin-2, whereas the most repressed genes were connective tissue growth factor, fibulin-3 and RGS-5. In addition, the full length coding sequence of two novel regulated capillary morphogenesis genes (CMGs) are presented. CMG-1 encodes a predicted intracellular 65 kDa protein with coiled-coil domains. A CMG-1-green fluorescent protein (GFP) chimera was observed to target to an intracellular vesicular compartment. A second novel gene, CMG-2, was found to encode a predicted intracellular protein of 45 kDa containing a transmembrane segment and a CMG-2-GFP chimera was observed to target to the endoplasmic reticulum. A recombinant portion of CMG-2 was found to bind collagen type IV and laminin, suggesting a potential role in basement membrane matrix synthesis and assembly. These data further elucidate the genetic events regulating capillary tube formation in a 3D matrix environment.

Quantitative Raman spectroscopy of highly fluorescent samples using pseudosecond derivatives and multivariate analysis.

Intense luminescence backgrounds cause significant problems in quantitative Raman spectroscopy, particularly in multivariate analysis where background suppression is essential. Taking second derivatives reduces the background, but differentiation increases the apparent noise that arises on spectra recorded with CCD detectors due to random, but fixed, variations in the pixel-to-pixel response. We have recently reported a very general method for correcting CCD fixed-pattern response in which spectra are taken at two or more slightly shifted spectrometer positions and are then subtracted to give a derivative-like shifted, subtracted Raman (SSR) spectrum. Here we show that differentiating SSR data (which has inherently higher S/N than the undifferenced data) yields spectra that are similar to those that are obtained from the normal two-step differentiation process and can be characterized as pseudo-second-derivative, PSD, spectra. The backgrounds are suppressed in the PSD spectra, which means they can be used directly in multivariate data analysis, but they have significantly higher S/N ratios than do simple second derivatives. To demonstrate the improvement brought about by using PSD spectra, we have analyzed known samples, consisting of simple binary mixtures of methanol and ethanol doped with laser dye. When the background levels of all samples included in the models were < or =10x greater than the intensity of the strongest Raman bands, partial least-squares calibration of the PSD data gave a standard error of prediction of 3.2%. Calibration using second derivatives gave a prediction error which was approximately twice as large, at 6.5%; however, when data with background levels . approximately 100x larger than the strongest Raman bands were included, the noise on the second-derivative spectra was so large as to prevent a meaningful calibration. Conversely, the PSD treatment of these samples gave a very satisfactory calibration with a standard error of prediction (3.3%) almost identical to that obtained when the most fluorescent samples were excluded. This method clearly has great potential for general purpose Raman analytical chemistry, because it does not depend on specialized equipment, is computationally undemanding, and gives stable and robust calibrations, even for samples in which the luminescence background level fluctuates between the extremes of being practically zero and completely dominating the Raman signal.

Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation.

B and T lymphocytes develop normally in mice lacking the guanine nucleotide exchange factor Vav-2. However, the immune responses to type II thymus-independent antigen as well as the primary response to thymus-dependent (TD) antigen are defective. Vav-2-deficient mice are also defective in their ability to switch immunoglobulin class, form germinal centers and generate secondary immune responses to TD antigens. Mice lacking both Vav-1 and Vav-2 contain reduced numbers of B lymphocytes and display a maturational block in the development of mature B cells. B cells from Vav-1(-/-)Vav-2(-/-) mice respond poorly to antigen receptor triggering, both in terms of proliferation and calcium release. These studies show the importance of Vav-2 in humoral immune responses and B cell maturation.

Disposable, stable media for reproducible surface-enhanced Raman spectroscopy.

Large numbers of identical and stable SE(R)RS [surface-enhanced (resonance) Raman]-active media, which are convenient to handle and manipulate but sufficiently inexpensive that they can be used once and then discarded, have been prepared by isolating nanoparticles from Ag and Au sols in hydrophilic polymer gels. The preparation simply involves mixing a suitable polymer with the sol to give a viscous suspension that can be coated onto a substrate and dried to form a hard translucent film. The films remain inactive until they are treated with aqueous analyte solution, which causes the film to swell and brings the analyte into contact with the active metal particles. The swollen films give strong SERS spectra which are effectively identical to those obtained from simple sols. The advantage of this method is that the dried polymers can be stored indefinitely before use and that they give a high degree of spectral reproducibility.

Composition profiling of seized ecstasy tablets by Raman spectroscopy.

Raman spectroscopy with far-red excitation has been investigated as a simple and rapid technique for composition profiling of seized ecstasy (MDMA, N-methyl-3,4-methylenedioxyamphetamine) tablets. The spectra obtained are rich in vibrational bands and allow the active drug and excipient used to bulk the tablets to be identified. Relative band heights can be used to determine drug/excipient ratios and the degree of hydration of the drug while the fact that 50 tablets per hour can be analysed allows large numbers of spectra to be recorded. The ability of Raman spectroscopy to distinguish between ecstasy tablets on the basis of their chemical composition is illustrated here by a sample set of 400 tablets taken from a large seizure of > 50,000 tablets that were found in eight large bags. The tablets are all similar in appearance and carry the same logo. Conventional analysis by GC-MS showed they contained MDMA. Initial Raman studies of samples from each of the eight bags showed that despite some tablet-to-tablet variation within each bag the contents could be classified on the basis of the excipients used. The tablets in five of the bags were sorbitol-based, two were cellulose-based and one bag contained tablets with a glucose excipient. More extensive analysis of 50 tablets from each of a representative series of sample bags have distribution profiles that showed the contents of each bag were approximately normally distributed about a mean value, rather than being mixtures of several discrete types. Two of the sorbitol-containing sample sets were indistinguishable while a third was similar but not identical to these, in that it contained the same excipient and MDMA with the same degree of hydration but had a slightly different MDMA/sorbitol ratio. The cellulose-based samples were badly manufactured and showed considerable tablet-to-tablet variation in their drug/excipient ratio while the glucose-based tablets had a tight distribution in their drug/excipient ratios. The degree of hydration in the MDMA feedstocks used to manufacture the cellulose-, glucose- and sorbitol-based tablets were all different from each other. This study, because it centres on a single seizure of physically similar tablets with the same active drug, highlights the fact that simple physical descriptions coupled with active drug content do not in themselves fully characterize the nature of the seized materials. There is considerable variation in the composition of the tablets within this single seizure and the fact that this variation can be detected from Raman spectra demonstrates that the potential benefits of obtaining highly detailed spectra can indeed translate into information that is not readily available from other methods but would be useful for tracing of drug distribution networks.

Rapid analysis of ecstasy and related phenethylamines in seized tablets by Raman spectroscopy.

Raman spectroscopy with far-red excitation has been used to study seized, tableted samples of MDMA (N-methyl-3,4-methylenedioxyamphetamine) and related compounds (MDA, MDEA, MBDB, 2C-B and amphetamine sulfate), as well as pure standards of these drugs. We have found that by using far-red (785 nm) excitation the level of fluorescence background even in untreated seized samples is sufficiently low that there is little difficulty in obtaining good quality data with moderate 2 min data accumulation times. The spectra can be used to distinguish between even chemically-similar substances, such as the geometrical isomers MDEA and MBDB, and between different polymorphic/hydrated forms of the same drug. Moreover, these differences can be found even in directly recorded spectra of seized samples which have been bulked with other materials, giving a rapid and non-destructive method for drug identification. The spectra can be processed to give unambiguous identification of both drug and excipients (even when more than one compound has been used as the bulking agent) and the relative intensities of drug and excipient bands can be used for quantitative or at least semi-quantitative analysis. Finally, the simple nature of the measurements lends itself to automatic sample handling so that sample throughputs of 20 samples per hour can be achieved with no real difficulty.

Examination of the physical state of chlorhexidine within viscoelastic, bioadhesive semisolids using raman spectroscopy.

This study examined the effects of polymeric components on the physical state of chlorhexidine within bioadhesive, semisolid formulations using Raman spectroscopy. Semisolid formulations were prepared in which chlorhexidine base (CHX, 5%w/w, particle size <63 microm) was dispersed in aqueous (phosphate-buffered saline, pH 6.8) polymer matrices consisting of one or more polymeric components, namely HEC (3%w/w), PVP (3%), and PC (PC, 3%). Raman spectra were recorded using 785-nm excitation and were typically accumulated for 360 s. The Raman spectra were dominated by the presence of CHX. The spectra of CHX in HEC and in HEC/PVP gels were indistinguishable from that for solid CHX as a result of the insolubility of CHX in these formulations. However, in systems containing PC and CHX, there was a shift in the strongest band from 1564 cm(-1) to 1608 cm(-1), which may be accredited to protonation of the basic CHX by the numerous carboxylic acidic groups on PC. Identical shifts in the band positions were observed when this protonation was modeled using ethanoic acid, supporting the view that there was a simple acid base reaction between PC and CHX. However, there were notable differences in the relative intensities of the peaks from these samples, with the spectrum of CHX in the PC matrix displaying properties intermediate between those of CHX dissolved in ethanoic acid and solid CHX diacetate. This may be accredited to the limited solubility of the CHX-PC ion pair. In matrices containing HEC and PC, no peak was observed at 1564 cm(-1), whereas the intensity of the peak at 1608 cm(-1) was increased. Therefore, in these formulations CHX was completely converted to the di-cation as a result of the synergistic effects of PC (which protonated CHX) and HEC (which solubilized the di-cation). In the absence of either HEC or PC, complete protonation was not achieved. It is suggested that this enhancement of solubility of H(2)CHX(2+) may be due to hydrogen bonding, given the hydroxylated nature of HEC. In conclusion, this study has shown the applicability of Raman spectroscopy for both the analysis of opaque, semisolid formulations and, additionally, for the examination of the state of therapeutic agents within such matrices. In particular, using Raman spectroscopy, it was uniquely possible to identify the roles of various polymeric components on both the ionization and solubilization of CHX within aqueous semisolid systems.

Identification of dyes on ancient Chinese paper samples using the subtracted shifted Raman spectroscopy method.

The Stein Collection in the British Library contains the Diamond Sutra, the world's oldest, dated, printed document. The paper of the Diamond Sutra and other documents from the Stein collection is believed to be dyed yellow by a natural extract, called huangbo, from the bark of Phellodendron amurense, which contains three major yellow chromophores: berberine, palmatine, and jatrorrhizine. Conservation of these documents requires definite information on the chemical composition of the dyes but no suitable, completely noninvasive analytical method is known. Here we report resonance Raman studies of a series of pure dyes, of plant materials and extracts, and of dyed ancient and modern paper samples. Resonance Raman spectroscopy is used to enhance the spectra of the dyes over the signals from the paper matrixes in which they are held. The samples all give resonance Raman spectra which are dominated by intense fluorescence, but by using SSRS (subtracted shifted Raman spectroscopy) we have obtained reliable spectra of the pure dyes, native bark from the Phellodendron amurense, modern paper dyed with huangbo extracted from this bark, and ancient paper samples. For both ancient paper samples whose pigment bands were detected, the relative intensities of the bands due to berberine and palmatine suggest that the ancient paper is richer in berberine than its modern counterpart. This is the first nondestructive in situ method for detection of these pigments in manuscripts, and as such has considerable potential benefit for the treatment of irreplaceable documents that are believed to be dyed with huangbo but documents on which conservation work cannot proceed without definite identification of the chemical compounds that they contain.