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Buyid Silk and the Tale of Bibi Shahrbanu: Identification of Biomarkers of Artificial Aging (Forgery) of Silk Mehdi Moini, and Christopher M. Rollman Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02854 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 18, 2017
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Analytical Chemistry
Buyid Silk and the Tale of Bibi Shahrbanu: Identification of Biomarkers of Artificial Aging (Forgery) of Silk Mehdi Moini*, Christopher M. Rollman George Washington University, Department of Forensic Sciences, Washington DC, USA ABSTRACT: Buyid silk forgery is one of the most famous silk forgeries in the world. In 1924-1925, excavation of the Bibi Shahrbanu site in Iran unearthed several silk textiles. The silks were thought to be of the Buyid period (934-1062 BCE) of the Persian Empire and have since been known as the “Buyid silks”. In the 1930s, more silk appeared and was reported as being from the Buyid period as well. Controversy over the authenticity of these silks escalated after the purchase of the silks by museums throughout the world. Extensive investigations of several of these silks have been conducted over the years with respect to iconography, weaving patterns, dyes/mordant, style and even radiocarbon dating. It was found that most of the silks are not from Buyid period. To test the authenticity of these silk fabrics, the recently developed silk dating technique using amino acid racemization (AAR) in conjunction with capillary electrophoresis mass spectrometry was applied to thirteen Buyid silk specimens from the Textile Museum collections. Among these silk specimens, the AAR ratios of only one specimen were consistent with authentic silk fabrics collected from various museums. In addition, the aspartic acid racemization ratio of this specimen was also consistent with its 14C dating. The other "Buyid silks" showed excessive levels of amino acid racemization not only for aspartic acid, but also for phenylalanine and tyrosine, inconsistent with racemization rates of these amino acids in authentic historical silk fabrics. Treatment of modern silk with a base at different pH and temperature reproduced the AAR pattern of the Buyid silks, implying that chemical treatment with a base at relatively high temperatures was perhaps the method used to artificially age these fabrics. The results imply that the racemization ratios of aspartic acid, phenylalanine, and tyrosine can be used as biomarkers for identification of naturally versus artificially aged silk.
Because of the limitations of radiocarbon (14C) dating for more recent objects (younger than 500 years old), the relatively large quantity (mg) of the silk needed for accelerator mass spectrometry (AMS 14C) dating, as well as the high cost of analysis, it is desirable to develop a new scientific technique for silk authentication. Racemization of various amino acids has been used as biomarkers for aging.5 In 2003, a capillary electrophoresis mass spectrometry (CE-MS) technique for separation and detection of amino acids and their D-, and L-isomers was developed and was later applied to amino acid racemization (AAR) dating of silk fabrics.6,7 By consuming only 10-20 µg of silk, and using relatively welldated silk specimens from various museums, a calibration curve was developed for dating silk ranging in age from modern to ~2500 years ago.7 Among 11 amino acid studied, aspartic acid (Asp) showed the fastest racemization rate, with a D/L = 0.45 for a ~2500 years old China’s Warring States silk.7 The purpose of this study was to develop a novel, rapid and sensitive technique for identification of silk forgery based on the CE-MS measurement of rate of racemization of various amino acids under natural and artificial aging and apply it to authentication of Buyid silks.
Introduction According to an Iranian folklore story1,2, Bibi Shahrbanu was a daughter of the last Sasanian king, Yazdegerd III (reigned 632-51). During Arab invasion of Iran, she was captured and taken to Medina, where she became the wife of Imam Hossain, son of Imam Alī, grandson of Prophet Muhammad. After the battle of Karbalāʾ (October 10, 680) she fled back to Iran, with her enemies in pursuit. Her enemies reached her at Ray (south of the current city of Tehran). In desperation, she tried to call on God; but instead of Yā hu (O God) her tongue slipped and she said Yā kūh (O mountain). The story is that, the mountain opened up and swallowed her; however, part of her vail (made of silk) remained unearthed and eventually became the current day Bibi Shahrbanu shrine near Ray, Iran. In 1924-1925, excavation of the Bibi Shahrbanu site unearthed several silk textiles, many of which were collected by private individuals. The silks were thought to be of the Buyid period of the Persian Empire and have since been known as the “Buyid silks”. In the 1930s, more silk appeared and was reported as being from the Buyid period as well. Controversy over the authenticity of these silks escalated after the purchase of the silks by museums throughout the world. Extensive investigations of several of these silks have been conducted over the years.3 It was found that most of the silk are not from Buyid period. One study found that radiocarbon dating results place many of the silks in 3 or 4 distinct age groups.4 Several silks were found to have been authentic and made between the 7th and 13th centuries AD. A second group of silks was dated between the 14th and 17th centuries AD, making them more recent than the Buyid period, but still of historical significance. The last two groups had dates from the 16th century to 1950 and definitively post-1950 (based on isotopes introduced from nuclear weapons), making these likely forgeries but made at different times.4
Methods and Instrumentation Sample Analysis CE-MS analysis of L- and D-amino acids in silk samples has been discussed before.7 Briefly, ~20 µg of silk specimens were digested in 6 N HCl at 110 °C for 2 hrs, dried in a centrifugal vacuum and then suspended in 0.1 N HCl for analysis. Racemization analyses were performed by capillary electrophoresis (Beckman-Coulter ProteomeLab™ PA 800; Fullerton, CA) mass spectrometry (Thermo LCQ Duo mass spectrometer, San Jose, CA) using underivatized fused-silica capillaries with 20 µm i.d., 150 µm o.d., ~110 cm in length
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with porous tips.8 Porous tip sheathless interface design was used to interface CE to MS using electrospray voltage of 1.1 kV. Analyses were performed using 30 mM (+)-(18-crown-6)2,3,11,12-tetracarboxylic acid (18-C-6-TCA) in water as a background electrolyte. All samples were injected using pressure injection (0.1 – 2 psi) and pressure-assisted CE separations occurred at 30 kV with an inlet pressure of 0.3 – 1 psi.9 Variations in separation of L- and D-amino acids are a result of minor variations in capillary length and separation conditions. The mass spectrometer heated capillary was set at 200 °C and a scan range of 505 – 800 m/z was used to detect the AA/18-C-6-TCA complexes. Under the experimental conditions used here, L-isomer of aspartic acid, phenylalanine, and tyrosine migrated before their D isomers.
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various amino acids. As an example, the electropherogram of the HCl digest of one of the specimens (TM 3.230) is shown in Figure S-2 (Bottom Panel) and it is compared with a modern undyed silk (Top Panel). Even though the Buyid silk fiber used for this experiment was green, the electropherograms were almost identical indicating that no amino acid or protein impurities were present. Of the 13 Buyid silk samples, only one (TM 73.663) was 14C dated to calibrated calendar age range with 95 percent confidence of 660-943 CE (or 1074-1357 years ago).4 The corresponding Asp racemization ratio for this specimen was ~30% (Figure 1 top panel) which according to our silk calibration curve translates to ~895 CE (1122 years old).7 Therefore, its aspartic acid racemization age is well within the 14C date. Another Buyid silk (TM 3.241) had a D/L-Asp age of ~1585 CE (432 years old). This age matches well with a 14C dated group of silks, which were passed off as being from the Buyid period (from Cleveland Museum of Art).4
Sample preparation for Artificial Aging Study An artificial aging study was performed using solutions of sodium carbonate and optima water (pH 8, 9, 11, and 13), which is conventionally used for silk degumming.10 The pH of the solutions was measured using a digital pH meter and adjusted with either 6 N HCl or 1 M NaOH to an actual pH of 8.03, 9.07, 11.03, 12.99. A “degumming” solution of just optima water (pH of 6.72) was also used. 2 mL of each solution were heated to 110 °C and 3 pieces (~1 cm squares) of modern non-degummed silk were placed into each solution. At the time intervals studied (30, 60, 120 min), one of the silk pieces was removed from the solution, washed in optima water, placed in a sample tube and dried at room temperature in an Eppendorf Vacufuge (New York, NY). For the water (pH 6.72), only the 120 min time interval was tested and for pH 13, the silk was completely dissolved in the degumming solution by 120 minutes. Updated Silk Calibration The previously published silk calibration curve based on aspartic acid racemization was updated with three new silk samples ranging in age from 500-1000 years old. These samples were added to the calibration curve, strengthening this portion of the curve, which was previously sparsely populated (Figure S-1). The inset of Figure S-1 are the extracted ion electropherograms for Asp (574 m/z) of selected silk samples showing the baseline separation of L- and D-Asp and the increase of D-Asp with age. The analysis of these new silk samples showed good concordance with our previously established calibration curve, indicating the robustness of the calibration curve. Table S-1 lists the silk samples used to generate Figure S-1 including the source of the silk, measured D/L (%) for that silk and the corresponding Asp racemization age of the samples.
Figure 1. Asp racemization for three 14C dated silk specimens including one authentic Buyid silk. However, the remaining forged Buyid silks that were analyzed had D/L-Asp age of much older than the Buyid dynasty (Figure 2). In addition to high D/L-Asp, the forged silks had very high D/L-Phe and D/L-Tyr (Figure 2), even higher than the authentic silk from China’s Warring States Dynasty period (BCE 475-221) (Figure 3). This was when the D/L-Phe and D/L-Tyr of Buyid silk TM 73.663 (14C dated to 1074-1357 CE) and Egyptian Tiraz TM 73.641 (14C dated to 993 CE) were almost zero (Figure 3). The results imply that a combination of D/L ratios for Asp, Phe, and Tyr could be used as markers for authentication of silk fibers.
Results and Discussion Buyid Silk Analysis The Buyid silk specimens obtained from the Textile Museum were analyzed to determine the AAR ratios for
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Analytical Chemistry
Figure 2. Extracted ion electropherograms showing the racemization of Asp (574 m/z), Phe (606 m/z), and Tyr (622 m/z) for three forged Buyid silks. The left peak for each AA is the L-isomer and the right peak is the D-isomer. observed after 60 mins (Figure 4, Panel D). Based on the calibration curve for silk, a 1 % increase in D-Asp is approximately equal to 50 years of aging. This means that when silk is exposed to degumming solution at pH 8.03, 9.07, 11.03, 12.99 (at 110 °C) for 1 hr, it is equivalent to 100, 400, 1700, and 3550 years of aging, respectively. Treating with a basic solution at 110 °C also resulted in racemization of other amino acids. Most notably, phenylalanine (Phe) and tyrosine (Tyr) had increased D-isomer formation over the time scales investigated here. Figure 5 shows racemization of Asp, Phe, and Tyr at pH 11 at various times. As shown after 120 minutes of degumming at 110 °C, the AAR profiles for Asp, Phe, and Tyr were very similar to the measured AAR ratios of the forged Buyid silks (Figure 2).
Artificial Aging Historically, the process of degumming silk used basic conditions at elevated temperatures. To mimic this process, raw, non-degummed silk was placed in solutions at pH 8.03, 9.07, 11.03, 12.99 at 110 °C for various times. As an example, Figure 4 shows the D/L Asp racemization in modern silk at pH 8.03, 9.07, 11.03, and 12.99 for 0 to 120 min at 110 °C.
Future Work While this study focused on the application of AA racemization to authentication of silk fabric, future studies will examine its utility to authentication of other proteinaceous relics including bone artifacts and will examine its use to study aging of historical objects due to environmental effects. Also, we recently introduced a portable CE and demonstrated its application to the analysis of amino acid racemization.11 In the future, this technology can be used with a suitable portable MS for on-site authentication of proteinaceous historical artifacts.
Figure 3. Extracted ion electropherograms showing the racemization of Asp (574 m/z), Phe (606 m/z) and Tyr (622 m/z) of three authentic silk samples including a carbon dated Buyid silk.
Conclusions Aspartic acid racemization in conjunction with a reliable calibration curve can be used to date silk specimens. D-Asp for one of oldest silks in museums (Warring States silk) is about ~45% of the L-aspartic acid. Even for ~2500 years old silk, the percentage of racemization of other amino acids
At pH 8.03, the %-D/L increased by ~4 % after 120 mins (Figure 4, Panel A). Increasing the pH to 9.07 resulted in a ~12 % increase in %-D/L Asp after 120 mins (Figure 4, Panel B). At pH 12.99, the silk sample was completely dissolved by 120 mins and an increase in %-D/L Asp of about 70 % was
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are minimal. For example, the D/L for Phe, which among the AAs studied had the second fastest racemization rate (after Asp) was only 5%. Silk fabrics, however, can be artificially aged using a basic solution at high temperature. The result is a silk fabric that looks very similar to a historical silk. However, forged silks can be identified by their excessively high racemization ratios not only for Asp, but more importantly for Phe and Tyr, which in historical silks are almost not existent. Therefore, we propose that amino acid racemization can be used as biomarkers for authentication of historical silks and for identification of silk forgery. Moreover, while 14C dating was able to place the Buyid silks in 3 or 4 distinct age groups and date silk specimens older than ~500 years old, the limitations of radiocarbon (14C) dating for more recent objects (younger than ~500 years old), the relatively large quantity (mg) of the silk needed and the high cost of analysis, makes the CE-MS an alternative method for authentication of historical silk. CE-MS has simple and fast sample preparation and analysis time, requires microgram quantities of silk (which makes it a minimally destructive technique), is relatively inexpensive, provides impurity check in conjunction with dating and provide authentication.
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Figure 5. Extracted ion electropherograms showing the effect of pH (11) and temperature (110 °C) at various times on racemization of Asp, Phe, and Tyr. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Document containing Figure S-1, Figure S-2 and Table S-1 (Supporting Information, PDF)
AUTHOR INFORMATION Corresponding Author *
[email protected] ACKNOWLEDGMENT We wish to thank the Textile Museums at the George Washington University, Washington DC for providing the Buyid Silk samples. We also wish to thank Dr. Sumru Krody, Senior Curator and Ms. Esther Methe, Senior Conservator (retired) at the Textile Museum, for their expertise and input regarding the Buyid silk samples. We also wish to thank Prof. Renata Holod of University of Pennsylvania and Zyezdana Dode of Russian Academy of Sciences for providing the new silk samples.
REFERENCES 1.
Boyce, M. Bulletin of the School of Oriental and African Studies, University of London 1967, 30(1), 30-44. 2. http://www.iranicaonline.org/articles/bibi-sahrbanu 3. Craddock, P. Scientific Investigation of Copies, Fakes and Forgeries, 1st ed.; Elsevier: New York, 2009. 4. Blair, S. S.; Bloom, J. M.; Wardwell, A. E. Ars Orientalis 1992, 22, 1-41. 5. Kalíková, K.; Šlechtová, T.; Tesarˇová E. Separations 2016, 3, 30-47. 6. Shultz, C. L.; Moini, M. Anal. Chem. 2003, 75(6), 15081513. 7. Moini, M.; Klauenberg, K.; Ballard, M. Anal. Chem. 2011, 83, 7577–7581. 8. Moini, M. Anal. Chem. 2007, 79, 4241-4246. 9. Cao, P.; Moini, M. Electrophoresis 1999, 19, 2200-2206. 10. Gulrajani, M.L. Rev. Prog. Coloration 1992, 22, 79-89. 11. Moini, M., Rollman C.M. J. Am. Soc. Mass Spectrom. 2016, 27, 388-393.
Figure 4. Asp racemization in modern silk at pH 8.03 (A), 9.07 (B), 11.03 (C), and 12.99 (D) at 110 °C for various times.
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Analytical Chemistry
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